Human genes, sequences and expression products

ABSTRACT

Human polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptides for therapeutic purposes. Antagonists against such polypeptides and their use as a therapeutic are also disclosed. Also disclosed are diagnostic methods for detecting disease which utilize the sequences and polypeptides.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Divisional of application Ser. No.09/417,540, filed Oct. 14, 1999, which is a Divisional of applicationSer. No. 08/705,771, filed Aug. 30, 1996 (now U.S. Pat. No. 6,054,289),which claims benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 60/002,993, filed Aug. 30, 1995.

BACKGROUND OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. The invention also relates toinhibiting the action of such polypeptides.

[0003] Identification and sequencing of human genes is a major goal ofmodern scientific research. For example, by identifying genes anddetermining their sequences, scientists have been able to make largequantities of valuable human “gene products.” These include humaninsulin, interferon, Factor VIII, tumor necrosis factor, human growthhormone, tissue plasminogen activator, and numerous other compounds.Additionally, knowledge of gene sequences can provide the key totreatment or cure of genetic diseases (such as muscular dystrophy andcystic fibrosis).

[0004] In accordance with one aspect of the present invention, there areprovided novel mature polypeptides, as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof. The polypeptides of the present invention are ofhuman origin.

[0005] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding the polypeptides,including mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs andbiologically active and diagnostically or therapeutically usefulfragments thereof.

[0006] In accordance with yet a further aspect of the present invention,there is provided a process for producing such polypeptides byrecombinant techniques comprising culturing recombinant prokaryoticand/or eukaryotic host cells, containing a nucleic acid sequence of thepresent invention, under conditions promoting expression of saidproteins and subsequent recovery of said proteins.

[0007] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptide for therapeutic and diagnosticpurposes.

[0008] In accordance with yet a further aspect of the present invention,there is also provided nucleic acid probes comprising nucleic acidmolecules of sufficient length to specifically hybridize to the nucleicacid sequences.

[0009] In accordance with yet a further aspect of the present invention,there are provided antibodies against such polypeptides.

[0010] In accordance with another aspect of the present invention, thereare provided agonists to the polypeptides.

[0011] In accordance with yet another aspect of the present invention,there are provided antagonists to such polypeptides, which may be usedto inhibit the action of such polypeptides, for therapeutic anddiagnostic purposes.

[0012] In accordance with still another aspect of the present invention,there are provided diagnostic assays for detecting diseases related tothe under-expression of the polypeptides of the present invention andmutations in the nucleic acid sequences encoding such polypeptides.

[0013] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

[0014] In the case where the polypeptides of the present invention arereceptors, there are provided processes for using the receptor to screenfor receptor antagonists and/or agonists and/or receptor ligands.

[0015] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0016] Table 1 sets forth information regarding identifyingpolynucleotide clone numbers, identification of the polynucleotidesequence which corresponds to the putative identification of thepolypeptide encoded by the polynucleotide, and cross-referencing to theSEQ ID NOS. as set forth in the sequence listing.

[0017] Table 2 includes information regarding identifying polypeptidenumbers, identification of the SEQ ID NOS. of the polypeptides, andcross-reference to the SEQ ID NO. which sets forth the amino acidsequence which corresponds to a given polypeptide in the sequencelisting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 shows the full length polynucleotide sequence (SEQ ID NO:1)of the HBGBA67X clone and correlates the coding region with the derivedamino acids (127 amino acids whose entire sequence (SEQ ID NO:12) isalso shown for the full length amyloid-like protein present in breasttissue.

[0019] FIGS. 2A-2B show the complete nucleotide (SEQ ID NO:2) and aminoacid sequence (SEQ ID NO:13) of the hADA2 gene and protein.

[0020] FIGS. 3A-3B show the full length sequence of the TFIId homologclone (SEQ ID NO:3) including the full length sequence of thepolynucleotide coding for TATA related factor (TRF) (SEQ ID NO:14).

[0021]FIG. 4 shows full length cDNA (SEQ ID NO:4) and deduced amino acidsequence (SEQ ID NO:15) of hRPB 11.

[0022] FIGS. 5A-5B show the full nucleotide sequence (SEQ ID NO:5) ofthe IRF3 gene and amino acid sequence (SEQ ID NO:16) resulting protein.The predicted molecular weight of IRF3 is 47,087; the predictedisoelectric is 5.06; and the net charge equals −14.

[0023]FIG. 6 shows individually the full length sequence (SEQ ID NO:6)of the TM4SF gene, the coding region sequence portion and the amino acidsequence (SEQ ID NO:17) of the translation product TM4SF.

[0024] FIGS. 7A-7B show the full length nucleotide sequence (SEQ IDNO:7) of TNFR AF1 C1, the complete coding sequence region of the fulllength sequence and the derived amino acid sequence (SEQ ID NO:18) ofthe resulting protein.

[0025]FIG. 8 shows the full length sequence (SEQ ID NO:8), the codingregion sequence and the derived amino acid sequence (SEQ ID NO:19) ofthe expression product protein of TM4SF (transmembrane 4 super family)CD53.

[0026]FIG. 9 shows the full length cDNA (SEQ ID NO:9) and the resultingexpression of the product protein (SEQ ID NO:20) of its coding regionfor retenoid receptor gamma.

[0027]FIG. 10 shows the full length nucleotide sequence (SEQ ID NO:10)(1237 bp) and the translation product (412 amino acid, SEQ ID NO:21)resulting from the nucleotide sequence for the cytosolic resiniferatoxinbinding protein RBP-26.

[0028]FIG. 11 shows the nucleotide sequence (SEQ ID NO:11) for the humanprotein (SEQ ID NO:22) kinase C inhibitor protein.

[0029] In accordance with an aspect of the present invention, there areprovided isolated nucleic acids (polynucleotides) which code for maturepolypeptides having the deduced amino acid sequences shown in the FIGS.1-11 or for the mature polypeptides encoded by the cDNA of the clonedeposited as ATCC Deposit No. 97242 on Aug. 15, 1995 with the ATCC,10801 University Boulevard, Manassas, Va. 20110-2209.

[0030] The polynucleotides of the present invention may be in the formof RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in SEQ ID NOS:1-11) or that ofthe deposited clone or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same mature polypeptide as the DNA of SEQ ID NOS:1-11or the deposited cDNA.

[0031] The polynucleotides which code for the mature polypeptides ofFIGS. 1-11 or for the mature polypeptides encoded by the deposited cDNAmay include, but are not limited to: only the coding sequence for themature polypeptide; the coding sequence for the mature polypeptide andadditional coding sequence such as a leader or secretory sequence or aproprotein sequence; the coding sequence for the mature polypeptide (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence (SEQ ID NO:1) 5′ and/or 3′ of the codingsequence (SEQ ID NO:2) for the mature polypeptide.

[0032] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0033] The present invention further relates to variants of thehereinabove described polynucleotides which code for fragments, analogsand derivatives of the polypeptide having the deduced amino acidsequences of FIGS. 1-11 or the polypeptides encoded by the cDNA of thedeposited clone. The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

[0034] Thus, the present invention includes polynucleotides encoding thesame mature polypeptides as shown in FIG. 1 or the same maturepolypeptides encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants code for a fragment,derivative or analog of the polypeptides of FIGS. 1-11 or thepolypeptides encoded by the cDNA of the deposited clone. Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

[0035] As hereinabove indicated, the polynucleotides may have a codingsequence which is a naturally occurring allelic variant of the codingsequences shown in FIGS. 1-11 or of the coding sequences of thedeposited clone. As known in the art, an allelic variant is an alternateform of a polynucleotide sequence which may have a substitution,deletion or addition of one or more nucleotides, which does notsubstantially alter the function of the encoded polypeptide.

[0036] The present invention also includes polynucleotides, wherein thecoding sequence for the mature polypeptide may be fused in the samereading frame to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also code for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

[0037] Thus, for example, the polynucleotide of the present inventionmay code for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence).

[0038] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al., Cell, 37:767 (1984)).

[0039] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0040] Fragments of the full length genes of the present invention maybe used as hybridization probes for a cDNA library to isolate the fulllength cDNA and to isolate other cDNAs which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype preferably have at least 30 bases and may contain, for example, 50or more bases. The probe may also be used to identify a cDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete gene including regulatory and promotorregions, exons and introns. An example of a screen comprises isolatingthe coding region of one of the genes by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or mRNA to determinewhich members of the library the probe hybridizes to.

[0041] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIGS. 1-11 (SEQ IDNOS:1-11) or the deposited cDNA(s).

[0042] Alternatively, the polynucleotides may have at least 20 bases,preferably 30 bases, and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which have anidentity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotides any of SEQ ID NOS:1-11, for example, forrecovery of the polynucleotide or as a diagnostic probe or as a PCRprimer.

[0043] Thus, the present invention is directed to polynucleotides havingat least a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to polynucleotides which encode the polypeptides ofSEQ ID NOS: 12-22, as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

[0044] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0045] The present invention further relates to polypeptides which havethe deduced amino acid sequence of SEQ ID NOS. 12-22 or which have theamino acid sequences encoded by the deposited cDNAs, as well asfragments, analogs and derivatives of such polypeptides.

[0046] The terms “fragment”, “derivative” and “analog” when referring tothe polypeptides of SEQ ID NOS. 12-22 or those encoded by the depositedcDNA, means polypeptides which retain essentially the same biologicalfunction or activity as such polypeptide. Thus, an analog and derivativeincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

[0047] The polypeptides of the present invention may be recombinantpolypeptides, natural polypeptides or synthetic polypeptides, preferablyrecombinant polypeptides.

[0048] The fragments, derivatives or analogs of the polypeptides of SEQID NOS. 12-22 or those encoded by the deposited cDNAs may be (i) thosein which one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, (ii) those in which one or moreof the amino acid residues includes a substituent group, (iii) those inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol) or (iv) those in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

[0049] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0050] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0051] The polypeptides of the present invention include thepolypeptides of SEQ ID NOS: 12-22 (in particular the maturepolypeptides) as well as polypeptides which have at least 70% similarity(preferably a 70% identity) to the polypeptides of SEQ ID NOS: 12-22 andmore preferably a 90% similarity (more preferably a 90% identity) to thepolypeptides of SEQ ID NOS: 12-22 and still more preferably a 95%similarity (still more preferably a 95% identity) to the individualpolypeptides of SEQ ID NOS: 12-22 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

[0052] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0053] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0054] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0055] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0056] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotides may be included in any one of a variety of expressionvectors for expressing the corresponding polypeptide. Such vectorsinclude chromosomal, nonchromosomal and synthetic DNA sequences, e.g.,derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeastplasmids; vectors derived from combinations of plasmids and phage DNA,viral DNA such as vaccinia, adenovirus, fowl pox virus, andpseudorabies. However, any other vector may be used as long as it isreplicable and viable in the host.

[0057] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0058] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda PL promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0059] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0060] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0061] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowesmelanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0062] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0063] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lac, lacZ, T3, T7, gpt, lambda PR, PL andtrp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0064] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, Dibner and Battey,Basic Methods in Molecular Biology, (1986)).

[0065] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0066] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0067] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0068] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0069] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0070] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0071] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0072] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0073] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0074] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0075] The polypeptides can be recovered and purified from recombinantcell cultures by methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

[0076] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

[0077] The amyloid-like gene and gene product may be employed as part ofa diagnostic process for the early detection of pre-cancerous growth orcancer in the breast. The amyloid protein forms amyloid fibrils which inturn are capable of attracting calcium molecules leading to calciumdeposition and calcification. Micro-fibrils and micro-calcificationcaused-by microinjury in the breast tissue result in densified breasttissue which is an early symptom detectable by mammography. Theamyloid-like protein gene according to the invention was isolated andrecovered as a full length gene by computer-assisted analysis ofexpression sequence tag data basis from a primary breast cancer library,a normal breast library, an activated monocyte library and an embryoniclibrary. The assembly of ESTs represents a full length gene which isillustrated in FIG. 1. Full length human ADA2 nucleotide sequence wasisolated from a 12 week old early stage human primary testes lambdalibrary.

[0078] The expression levels of the amyloid-like protein according tothe invention may be detectable in the serum and/or ductal fluid of thebreast due to its secretory nature, thus the amyloid-like protein may beemployed as a target for detection in such breast fluids. Further,examination of tissue samples from the breast for increased levels ofthe amyloid protein according to the invention may be helpful as part ofan overall diagnostic regimen to screen for abnormal breast tissuegrowth or for breast cancer.

[0079] The amyloid-like protein according to the invention is toxic tosurrounding breast cells which leads to apoptosis. The deposition ofthis protein in the breast tissue may be an early lesion for cancerousgrowth in the breast. Thus, this gene may be a target for breast cancerdiagnosis.

[0080] Human transcriptional regulator hADA2 is the human homolog of ayeast factor identified as being important for mediating thetranscriptional activation properties of the Herpes Simplextransactivator VP16. It is possible that being able to control theactivity of this factor (perhaps through anti-sense or screenedantagonists) will allow the regulation of specific viral and human geneswhose expression is controlled by this factor. This could lead to thecontrolled regulation of certain medically important genes. Furthermore,it is possible that disruption of this gene could result in unregulatedtranscription leading to cancer, in which case gene therapy would bemedically important. Administration of HADA2 via gene therapy may beemployed to treat cancer since disruption of the HADA2 gene results inunregulated transcription. We have recently mapped the chromosomallocation of this gene to 17q12-21. The gene encoding the HADA2 proteinwas isolated from a 12 week old human primary testes library.

[0081] Modulating the activity of the human transcription regulatorHADA2 may be employed to enhance or reduce the amount of a particulargene product produced. For example, in the case of an elevated level ofa polypeptide the gene responsible may be down-regulated by inhibitingHADA2. Likewise, if an up-regulation of a gene product is desired, e.g.,growth hormone, HADA2 may be stimulated.

[0082] Human transcription regulator factor (hTRF) is a homolog of theTATA Box Binding protein which plays a pivotal role in the expression ofall genes. The full length cDNA of TRF was isolated by screening a humantestes library. The hRPB 11 gene was isolated from a subtracted humanpituitary library. It is possible that lack or overexpression of thisgene could lead to unregulated transcription leading to cancer. Thehuman transcription factor hTRF may play a pivotal role in theexpression of nearly all human genes since it is thought to bind to the“TATA box” upstream of all translated genes. Accordingly, modulation ofhTRF, via gene therapy, stimulation and antagonism may be employed tocontrol gene expression. Lack of hTRF may cause unregulatedtranscription which may lead to cancer. Accordingly administration ofhTRF protein, or administration of the hTRF gene via gene therapy may beemployed to treat cancer.

[0083] The human RNA polymerase subunits hRPB8, hRPB 10 and hRPB11 playvital roles in mRNA synthesis since they possess the catalytic machineryfor the formation of the 3′-5′ phosphodiester bonds betweenribonucleoside triphosphates and respond to signals from the multiplefactors involved in regulating their function during initiation andelongation of mRNA synthesis. These subunits are able to support normalyeast cell growth in vivo. The coding region in some flanking 5′ and 3′UTR have been sequenced. The protein has a predicted molecular weight of13,293; an isoelectric point of 5.73 and is 117 residues long.

[0084] Accordingly, since the subunits are vital to mRNA synthesis,their administration may be employed to up-regulate the expression ofcertain genes and to down-regulate others as needed. Administration maybe via gene therapy. Abnormal cellular proliferation, e.g., cancer, maybe treated with the subunits since lack of expression of these genes maylead to unregulated transcription.

[0085] The human interferon regulatory factor IRF3 gene shows stronghomology to a group of transcription factors including IRF1 (InterferonRegulatory factor 1) and IRF2 (interferon Regulatory factor 2) which areimportant in mediating the transcriptional activation ofinterferon-alpha and -beta induced genes. It is possible that this genealso is important in mediating the transcriptional activation propertiesof interferon and that this factor may have some of the propertiesassociated with interferon such as anti-viral activity. The humaninterferon regulatory factor IRF3 is potentially important in regulatingthe transcriptional activation of interferon-α and -β genes. IRF3 mayalso be important in mediating the transcriptional activation propertiesof interferon. The IRF3 polypeptide may be employed as an anti-viralagent. The administration of the IRF3 gene and its gene product may beemployed to enhance the expression of interferon which has manymedically important uses. The IRF3 gene was isolated from a human adultretina library.

[0086] The TM4SF gene may be employed as a target for the development ofcompounds to treat human T-cell leukemia virus type I since severalmonoclonal-antibodies inhibitory to syncytium formation targeted thisTM4SF molecule.

[0087] The TM4SF gene may also be employed in the regulation of cellgrowth. This gene may also be employed as an immunogen or target toimplement active and passive immunotherapy in patients with cancer. Thegene encoding TM4SF was isolated from a human T-cell lymphoma library.

[0088] The TNFR-AF1, C1 gene and gene product may be employed toregulate B-lymphocyte proliferation, immunoglobulin class-switching andapoptosis. The TNFR-AF1 may also be employed to up-regulate thebiological activity of TNF which is known to regress tumors. The geneencoding TNFR-AF1 C1 was isolated from an activated human nutrophillibrary.

[0089] The TM4SF, CD53 gene and gene product may be employed to regulatelymphoma cell growth and may also be employed to regulate cell growth.The gene encoding TM4SF (transmembrane 4 super family), CD53 wasisolated from a human tumor pancreas library.

[0090] The retinoid X receptor y may be employed to treat psoriasis andrecalcitrincystic acne and cancer. This retinoid X receptor y may alsobe employed to prevent a variety of pre-malignant lesions of skin andmucous membranes. The receptor may also be employed as a tumorsuppressor. The receptor may also be employed to stimulate cellproliferation, differentiation and keratinization. The receptor may alsobe employed to treat X linked adrenal hypoplasia and hypogonatropichypoglonatism. The gene encoding retinoid X receptor gamma was isolatedfrom a human fetal lung III library.

[0091] The cytosolic resiniferatoxin binding protein (RBP-26) may beemployed to reduce pain sensation due to its ability to selectivelyblock mechanoheat nociceptors and warm receptors of the skin that areknown to play a significant role in sensation of pain. The gene encodingRBP-26 was isolated from a human osteoclastoma stromal cell library.

[0092] The protein kinase C inhibitor protein has siginificant medicalapplication uses such as inhibiting tumor cell growth and in regulatingthe many physiological functions that are mediated by the activation ofprotein kinase C. The gene encoding the protein kinase C inhibitorprotein was isolated from a human corpus colosum library.

[0093] The polynucleotides and polypeptides of the present invention maybe employed as research reagents and materials for discovery oftreatments and diagnostics to human disease.

[0094] This invention provides a method for identification of thereceptors for the polypeptides listed in Table 1. The gene encoding thereceptor can be identified by numerous methods known to those of skillin the art, for example, ligand panning and FACS sorting (Coligan, etal., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the respective polypeptide, and a cDNA librarycreated from this RNA is divided into pools and used to transfect COScells or other cells that are not responsive to the proteins.Transfected cells which are grown on glass slides are exposed to labeledprotein. The protein can be labeled by a variety of means includingiodination or inclusion of a recognition site for a site-specificprotein kinase. Following fixation and incubation, the slides aresubjected to auto-radiographic analysis. Positive pools are identifiedand sub-pools are prepared and re-transfected using an iterativesub-pooling and re-screening process, eventually yielding a single clonethat encodes the putative receptor.

[0095] As an alternative approach for receptor identification, labeledprotein can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the protein-receptor can be excised, resolved into peptidefragments, and subjected to protein microsequencing. The amino acidsequence obtained from microsequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

[0096] This invention provides a method of screening compounds toidentify those which enhance (agonists) or block (antagonists)interaction of protein to receptor. An agonist is a compound whichincreases the natural biological functions, while antagonists eliminatesuch functions. As an example, a mammalian cell or membrane preparationexpressing the receptor would be incubated with labeled protein in thepresence of the drug. The ability of the drug to enhance or block thisinteraction could then be measured.

[0097] Alternatively, the response of a known second messenger systemfollowing interaction of protein and receptor would be measured andcompared in the presence or absence of the drug. Such second messengersystems include but are not limited to, cAMP guanylate cyclase, ionchannels or phosphoinositide hydrolysis.

[0098] In the case where the polypeptides of the present invention arereceptor polypeptides, the present invention also relates to methods fordetermining whether a ligand can bind to the receptor which comprisestransfecting a cell population (one presumed not to contain a receptor)with the appropriate vector expressing the receptor, such that the cellwill now express the receptor. A suitable response system is obtained bytransfection of the DNA into a suitable host containing the desiredsecond messenger pathways including cAMP, ion channels, phosphoinositidekinase, or calcium response. Such a transfection system provides aresponse system to analyze the activity of various ligands exposed tothe cell. Ligands chosen could be identified through a rational approachby taking known ligands that interact with similar types of receptors orusing small molecules, cell supernatants or extracts or naturalproducts.

[0099] The present invention also relates to an assay for identifyingpotential antagonists. An example of such an assay combines the proteinand a potential antagonist with membrane-bound receptors or recombinantreceptors under appropriate conditions for a competitive inhibitionassay. The protein can be labeled, such as by radio activity, such thatthe number of molecules bound to the receptor can determine theeffectiveness of the potential antagonist.

[0100] The polypeptides listed in Table 1 of the present invention whichhave putatively been identified as receptors may be employed in aprocess for screening for antagonists and/or agonists for the receptor.

[0101] In general, such screening procedures involve providingappropriate cells which express the receptor on the surface thereof. Inparticular, a polynucleotide encoding the receptor of the presentinvention is employed to transfect cells to thereby express thereceptor. Such transfection may be accomplished by procedures ashereinabove described.

[0102] One such screening procedure involves the use of melanophoreswhich are transfected to express the receptor of the present invention.Such a screening technique is described in PCT WO 92/01810 publishedFeb. 6, 1992.

[0103] Thus, for example, such assay may be employed for screening for areceptor antagonist by contacting the melanophore cells which encode thereceptor with both the receptor ligand and a compound to be screened.Inhibition of the signal generated by the ligand indicates that acompound is a potential antagonist for the receptor, i.e., inhibitsactivation of the receptor.

[0104] The screen may be employed for determining an agonist bycontacting such cells with compounds to be screened and determiningwhether such compound generates a signal, i.e., activates the receptor.

[0105] Other screening techniques include the use of cells which expressthe receptor (for example, transfected CHO cells) in a system whichmeasures extracellular pH changes caused by receptor activation, forexample, as described in Science, volume 246, pages 181-296 (October1989). For example, potential agonists or antagonists may be contactedwith a cell which expresses the receptor and a second messengerresponse, e.g. signal transduction or pH changes, may be measured todetermine whether the potential agonist or antagonist is effective.

[0106] Another such screening technique involves introducing RNAencoding the receptor into xenopus oocytes to transiently express thereceptor. The receptor oocytes may then be contacted in the case ofantagonist screening with the receptor ligand and a compound to bescreened, followed by detection of inhibition of a calcium signal.

[0107] Another screening technique involves expressing the receptor inwhich the receptor is linked to a phospholipase C or D. Asrepresentative examples of such cells, there may be mentionedendothelial cells, smooth muscle cells, embryonic kidney cells, etc. Thescreening for an antagonist or agonist may be accomplished ashereinabove described by detecting activation of the receptor orinhibition of activation of the receptor from the phospholipase secondsignal.

[0108] Another method involves screening for antagonists by determininginhibition of binding of labeled ligand to cells which have the receptoron the surface thereof. Such a method involves transfecting a eukaryoticcell with DNA encoding the receptor such that the cell expresses thereceptor on its surface and contacting the cell with a potentialantagonist in the presence of a labeled form of a known ligand. Theligand can be labeled, e.g., by radioactivity. The amount of labeledligand bound to the receptors is measured, e.g., by measuringradioactivity of the receptors. If the potential antagonist binds to thereceptor as determined by a reduction of labeled ligand which binds tothe receptors, the binding of labeled ligand to the receptor isinhibited.

[0109] The present invention also provides a method for determiningwhether a ligand not known to be capable of binding to a receptor canbind to such receptor which comprises contacting a mammalian cell whichexpresses a receptor with the ligand under conditions permitting bindingof ligands to the receptor, detecting the presence of a ligand whichbinds to the receptor and thereby determining whether the ligand bindsto the receptor. The systems hereinabove described for determiningagonists and/or antagonists may also be employed for determining ligandswhich bind to the receptor.

[0110] In general, antagonists for receptors which are determined byscreening procedures may be employed for a variety of therapeuticpurposes. For example, such antagonists have been employed for treatmentof hypertension, angina pectoris, myocardial infarction, ulcers, asthma,allergies, psychoses, depression, migraine, vomiting, and benignprostatic hypertrophy.

[0111] Agonists for receptors are also useful for therapeutic purposes,such as the treatment of asthma, Parkinson's disease, acute heartfailure, hypotension, urinary retention, and osteoporosis.

[0112] Potential antagonists against the polypeptides of the presentinvention include an antibody, or in some cases, an oligopeptide, whichbinds to the polypeptide. Alternatively, a potential antagonist may be aclosely related protein which binds to the receptors of the polypeptide,however, they are inactive forms of the polypeptide and thereby inhibitthe action of the polypeptides.

[0113] Another potential antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5′ coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al,Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),thereby preventing transcription and the production of the polypeptide.The antisense RNA oligonucleotide hybridizes to the mRNA in vivo andblocks translation of the mRNA molecule into the polypeptide(Antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of the protein.

[0114] Potential antagonists include a small molecule which binds to andoccupies the active site of the polypeptide or to the receptor where thepolypeptide of the present invention is a receptor, thereby making itinaccessible to substrate such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall peptides or peptide-like molecules.

[0115] Another potential antagonist includes a soluble form of thereceptor polypeptides, e.g. a fragment of the receptor, which binds tothe ligand and prevents the ligand from interacting with membrane boundreceptors.

[0116] Antagonists to the human transcription regulator hADA2 may beemployed to regulate the expression of Herpes simplex transactivatorVP16, since hADA2 mediates its transcriptional activation properties.Many medically important genes may also be regulated by the antagonismof hADA2.

[0117] Antagonists to TATA related factor (TRF) may be employed tocontrol general protein expression and for the regulation of theexpression of specific important gene groups.

[0118] Antagonists to RNA polymerase subunits HRPB8, HRPB10 and HRPB11may be employed to treat cancer since over expression of these subunitsmay lead to unregulated transcription.

[0119] Antagonists to interferon related factor-3 (IRF-3) may beemployed to down regulate the overexpression of interferon with itsadverse effects.

[0120] Antagonists to TM4SF may be employed to inhibit tumor growth.

[0121] Antagnoists to TNFR AF1, C1 may be employed to inhibitinflammation and apoptosis.

[0122] Antagnoists to TM4SF (transmembrane 4 super family) CD53 may beemployed to inhibit certain leukemias.

[0123] Antagonists to the retinoid X receptor y may be employed to treatpsoriasis and inflammation. The antagonists may also be employed toprevent and/or treat hyperplasia and tumors in the lung, breast andother tissues.

[0124] Antagonists to protein kinase C inhibitor protein may be employedto inhibit the activation function of protein kinase C.

[0125] The antagonists may be employed therapeutically in a compositionwith a pharmaceutically acceptable carrier, e.g., as hereinafterdescribed.

[0126] The polypeptides of the present invention and agonists andantagonists may be employed in combination with a suitablepharmaceutical carrier. Such compositions comprise a therapeuticallyeffective amount of the polypeptide or agonist or antagonist, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

[0127] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides of the present invention or agonists orantagonists may be employed in conjunction with other therapeuticcompounds.

[0128] The pharmaceutical compositions may be administered in aconvenient manner such as by the oral, topical, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, they are administered in an amount of at leastabout 10 μg/kg body weight and in most cases they will be administeredin an amount not in excess of about 8 mg/kg body weight per day. In mostcases, the dosage is from about 10 μg/kg to about 1 mg/kg body weightdaily, taking into account the routes of administration, symptoms, etc.

[0129] The polypeptides and agonists and antagonists which arepolypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as “gene therapy.”

[0130] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art and are apparentfrom the teachings herein. For example, cells may be engineered by theuse of a retroviral plasmid vector containing RNA encoding a polypeptideof the present invention.

[0131] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Forexample, a packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

[0132] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0133] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller et al., Biotechniques, 7(9):980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B 19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

[0134] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0135] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PES01,PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy, 1:5-14 (1990), which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

[0136] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0137] This invention is also related to the use of the DNA (RNA)sequences diagnostically. Detection of a mutated form of sequences willallow a diagnosis of a disease or a susceptibility to a disease whichresults from under-expression of the protein.

[0138] Individuals carrying mutations in a human gene of the presentinvention may be detected at the DNA level by a variety of techniques.Nucleic acids for diagnosis may be obtained from a patient's cells, suchas from blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose. Asan example, PCR primers complementary to the nucleic acid encoding theprotein can be used to identify and analyze mutations. For example,deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to radiolabeled RNA oralternatively, radiolabeled antisense DNA sequences. Perfectly matchedsequences can be distinguished from mismatched duplexes by RNase Adigestion or by differences in melting temperatures.

[0139] Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

[0140] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

[0141] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0142] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0143] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0144] The present invention also relates to a diagnostic assay fordetecting altered levels of the polypeptides of the present inventionand soluble form of the receptor polypeptides of the present invention,in various tissues since an over-expression of the proteins compared tonormal control tissue samples can detect the presence of a disease.Assays used to detect levels of protein in a sample derived from a hostare well-known to those of skill in the art and includeradioimmunoassays, competitive-binding assays, Western Blot analysis andpreferably an ELISA assay. An ELISA assay initially comprises preparingan antibody specific to the antigen, preferably a monoclonal antibody.In addition a reporter antibody is prepared against the monoclonalantibody. To the reporter antibody is attached a detectable reagent suchas radioactivity, fluorescence or in this example a horseradishperoxidase enzyme. A sample is now removed from a host and incubated ona solid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein such as bovine serum albumin.Next, the monoclonal antibody is incubated in the dish during which timethe monoclonal antibodies attach to any proteins attached to thepolystyrene dish. All unbound monoclonal antibody is washed out withbuffer. The reporter antibody linked to horseradish peroxidase is nowplaced in the dish resulting in binding of the reporter antibody to anymonoclonal antibody bound to the protein of interest. Unattachedreporter antibody is then washed out. Peroxidase substrates are thenadded to the dish and the amount of color developed in a given timeperiod is a measurement of the amount of protein present in a givenvolume of patient sample when compared against a standard curve.

[0145] A competition assay may be employed wherein antibodies specificto the protein is attached to a solid support and labeled protein and asample derived from the host are passed over the solid support and theamount of label detected attached to the solid support can be correlatedto a quantity of the protein in the sample.

[0146] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0147] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region of the gene is used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0148] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0149] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAhaving at least 50 or 60 bases. For a review of this technique, seeVerma et al., Human Chromosomes: a Manual of Basic Techniques, PergamonPress, New York (1988).

[0150] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0151] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0152] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0153] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0154] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0155] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,Nature, 256:495-497, 1975), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today 4:72, 1983), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

[0156] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0157] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0158] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0159] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbases, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0160] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0161] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0162] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0163] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0164] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLE 1

[0165] Bacterial Expression and Purification of the Proteins

[0166] The DNA sequence encoding any of the proteins, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′sequences of the processed protein (minus the signal peptide sequence)and the vector sequences 3′ to the gene. Additional nucleotidescorresponding to the DNA sequence are added to the 5′ and 3′ sequencesrespectively. The 5′ oligonucleotide primer may contain, for example, arestriction enzyme site followed by nucleotides of coding sequencestarting from the presumed terminal amino acid of the processed protein.The 3′ sequence may, for example, contain complementary sequences to arestriction enzyme site and also be followed by nucleotides of thenucleic acid sequence encoding the protein of interest. The restrictionenzyme sites correspond to the restriction enzyme sites on a bacterialexpression vector, for example, pQE-9 (Qiagen, Inc. Chatsworth, Calif.).pQE-9 encodes antibiotic resistance (Ampr), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-9 is then digested with the restriction enzymes corresponding torestriction enzyme sites contained in he primer sequences. The amplifiedsequences are ligated into pQE-9 and inserted in frame with the sequenceencoding for the histidine tag and the RBS. The ligation mixture is thenused to transform an E. coli strain, for example, M15/rep 4 (Qiagen) bythe procedure described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory Press, (1989). M15/rep4contains multiple copies of the plasmid pREP4, which expresses the lacIrepressor and also confers kanamycin resistance (Kan^(r)). Transformantsare identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis. Clones containing thedesired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation. The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedprotein is purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography411:177-184 (1984)). The protein is eluted from the column in 6 molarguanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione(reduced) and 2 mmolar glutathione (oxidized). After incubation in thissolution for 12 hours the protein is dialyzed to 10 mmolar sodiumphosphate.

EXAMPLE 2

[0167] Cloning and Expression of the Proteins Using the BaculovirusExpression System

[0168] The DNA sequence encoding one of the full length proteins, isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ sequences of the gene.

[0169] The 5′ primer may contain a restriction enzyme site and befollowed by a number of nucleotides resembling an efficient signal forthe initiation of translation in eukaryotic cells (Kozak, J. Mol. Biol.,196:947-950 (1987) which is just behind the first few nucleotides of thegene of interest.

[0170] The 3′ primer may also contain a restriction endonuclease andhave extra nucleotides which are complementary to the 3′ non-translatedsequence of the gene. The amplified sequences are isolated from a 1%agarose gel using a commercially available kit (“Geneclean,” BIO 101Inc., La Jolla, Calif.). The fragment is then digested with theendonucleases and purified again on a 1% agarose gel. This fragment isdesignated F2.

[0171] A vector, for example, pA2 or pRG1 (modification of pVL941vector, discussed below) may be used for the expression of the proteinusing the baculovirus expression system (for review see: Summers, M. D.and Smith, G. E. 1987, A manual of methods for baculovirus vectors andinsect cell culture procedures, Texas Agricultural Experimental StationBulletin No. 1555). These vectors contain the strong polyhedrin promoterof the Autographa californica nuclear polyhedrosis virus (AcMNPV)followed by the recognition sites for the respective restrictionendonucleases. The polyadenylation site of the simian virus (SV)40 isused for efficient polyadenylation. For an easy selection of recombinantvirus the beta-galactosidase gene from E. coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

[0172] The plasmid is digested with the restriction enzymes anddephosphorylated using calf intestinal phosphatase by procedures knownin the art. The DNA is then isolated from a 1% agarose gel using thecommercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).This vector DNA is designated V2.

[0173] Fragment F2 and the dephosphorylated plasmid V2 are ligated withT4 DNA ligase. An E. coli strain, for example, HB101 cells are thentransformed and bacteria which contain the recombinant plasmid areidentified using the restriction enzymes. The sequence of the clonedfragment is confirmed by DNA sequencing.

[0174] 5 μg of the plasmid is co-transfected with 1.0 μg of acommercially available linearized baculovirus (“BaculoGold™ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

[0175] 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid are mixedin a sterile well of a microtiter plate containing 50 μl of serum freeGrace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards10 μl Lipofectin plus 90 μl Grace's medium are added, mixed andincubated for 15 minutes at room temperature. Then the transfectionmixture is added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate is rocked back and forth to mix the newly addedsolution. The plate was then incubated for 5 hours at 27° C. After 5hours the transfection solution is removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum is added.The plate is put back into an incubator and cultivation continued at 27°C. for four days.

[0176] After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0177] Four days after the serial dilution the virus is added to thecells and blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculovirus is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

[0178] Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus at a multiplicity of infection (MOI) of 2. Six hours laterthe medium is removed and replaced with SF900 II medium minus methionineand cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5μCi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham) are added. Thecells are further incubated for 16 hours before they are harvested bycentrifugation and the labelled proteins visualized by SDS-PAGE andautoradiography.

EXAMPLE 3

[0179] Expression of Recombinant Protein in COS Cells

[0180] The expression of plasmid, protein-HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E. coli replication origin, 4) CMVpromoter followed by a polylinker region, an SV40 intron andpolyadenylation site. A DNA fragment encoding the entire precursor and aHA tag fused in frame to its 3′ end is cloned into the polylinker regionof the vector, therefore, the recombinant protein expression is directedunder the CMV promoter. The HA tag corresponds to an epitope derivedfrom the influenza hemagglutinin protein as previously described (I.Wilson, H. Niman, R. Heighten, A Cherenson, Connolly, and Lerner, Cell37:767, (1984)). The infusion of HA tag to the target protein allowseasy detection of the recombinant protein with an antibody thatrecognizes the HA epitope.

[0181] The DNA sequence encoding the protein is constructed by PCR usingtwo primers, a 5′ primer containing a restriction enzyme site followedby a number of nucleotides of the coding sequence starting from theinitiation codon, and a 3′ primer also containing complementarysequences to a restriction site, translation stop codon, HA tag and thelast few nucleotides of the coding sequence (not including the stopcodon). Therefore, the PCR product contains restriction enzyme sites,coding sequence followed by HA tag fused in frame, a translationtermination stop codon next to the HA tag, and the other restrictionenzyme site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp,are digested with appropriate restriction enzymes and ligated. Theligation mixture is transformed into an E. coli strain, for example,SURE (Stratagene Cloning Systems, La Jolla, Calif.) and the transformedculture is plated on ampicillin media plates and resistant colonies areselected. Plasmid DNA is isolated from transformants and examined byrestriction analysis for the presence of the correct fragment. Forexpression of the recombinant protein, COS cells are transfected withthe expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch,T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold SpringLaboratory Press, (1989)). The expression of the HA protein is detectedby radiolabelling and immunoprecipitation method (E. Harlow, D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Cells are labelled for 8 hours with 35 S-cysteine two days posttransfection. Culture media is then collected and cells are lysed withdetergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5%DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Bothcell lysate and culture media are precipitated with an HA specificmonoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGEgels.

EXAMPLE 4

[0182] Isolation of a Selected Clone from the Deposited cDNA Library

[0183] Two approaches are used to isolate a particular gene out of thedeposited cDNA library.

[0184] In the first, a clone is isolated directly by screening thelibrary using an oligonucleotide probe. To isolate a particular gene, aspecific oligonucleotide with 30-40 nucleotides is synthesized using anApplied Biosystems DNA synthesizer according to a fragment of the genesequence. The oligonucleotide is labeled with 32 P-ATP using T4polynucleotide kinase and purified according to the standard protocol(Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, Cold Spring, N.Y., 1982). The Lambda cDNA librarydeposited is plated on 1.5% agar plate to the density of 20,000-50,000pfu/150 mm plate. These plates are screened using Nylon membranesaccording to the standard phage screening protocol (Stratagene, 1993).Specifically, the Nylon membrane with denatured and fixed phage DNA isprehybridized in 6×SSC, 20 mM NaH₂ PO₄, 0.4% SDS, 5× Denhardt's 500μg/ml denatured, sonicated salmon sperm DNA; and 6×SSC, 0.1% SDS. Afterone hour of prehybridization, the membrane is hybridized withhybridization buffer 6×SSC, 20 mM NaH₂ PO₄, 0.4% SDS, 500 ug/mldenatured, sonicated salmon sperm DNA with 1×10 cpm/ml ³²P-probeovernight at 42° C. The membrane is washed at 45-50° C. with washingbuffer 6×SSC, 0.1% SDS for 20-30 minutes dried and exposed to KodakX-ray film overnight. Positive clones are isolated and purified bysecondary and tertiary screening. The purified clone is sequenced toverify its identity to the fragment sequence.

[0185] An alternative approach to screen the deposited cDNA library isto prepare a DNA probe corresponding to the entire sequence. To preparea probe, two oligonucleotide primers of 17-20 nucleotides derived fromboth ends of the sequence are synthesized and purified. These twooligonucleotide are used to amplify the probe using the cDNA librarytemplate. The DNA template is prepared from the phage lysate of thedeposited cDNA library according to the standard phage DNA preparationprotocol (Maniatis et al.). The polymerase chain reaction is carried outin 25 μl of reaction mixture with 0.5 ug of the above cDNA template. Thereaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 μM each ofdATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taqpolymerase. Thirty five cycles of PCR (denaturation at 94° C. for 1 min;annealing at 55° C. for 1 min; elongation at 72° C. for 1 min) areperformed with the Perkin-Elmer Cetus automated thermal cycler. Theamplified product is analyzed by agarose gel electrophoresis and the DNAband with expected molecular weight is excised and purified. The PCRproduct is verified to be the probe by subcloning and sequencing the DNAproduct. The probe is labeled with the Multiprime DNA Labelling System(Amersham) at a specific activity <1×10⁹ dpm/μg. This probe is used toscreen the deposited lambda cDNA library according to Stratagene'sprotocol. Hybridization is carried out with 5X TEN (20XTEN:0.3M Tris-HClpH 8.0, 0.02M EDTA and 3M NaCl), 5× Denhardts, 0.5% sodiumpyrophosphate, 0.1% SDS, 0.2 mg/ml heat denatured salmon sperm DNA and1×10⁶ cpm/ml of [³²P]-labeled probe at 55 C. for 12 hours. The filtersare washed in 0.5× TEN at room temperature for 20-30 min., then at 55°C. for 15 min. The filters are dried and autoradiographed at −70° C.using Kodak XAR-5 film. The positive clones are purified by secondaryand tertiary screening. The sequence of the isolated clone are verifiedby DNA sequencing.

[0186] General procedures for obtaining complete sequences from probesare summarized as follows:

[0187] Procedure

[0188] Selected human DNA from a probe corresponding to part of thehuman gene is purified e.g., by endonuclease digestion using EcoR1, gelelectrophoresis, and isolation of the probe sequence by removal from lowmelting agarose gel. The isolated insert DNA, is radiolabeled e.g., with³²P labels, preferably by nick translation or random primer labeling.The labeled probe insert is used as a probe to screen a lambda phagecDNA library or a plasmid cDNA library. Colonies containing genesrelated to the probe cDNA are identified and purified by knownpurification methods. The ends of the newly purified genes arenucleotide sequenced to identify full length sequences. Completesequencing of full length genes is then performed by Exonuclease IIIdigestion or primer walking. Northern blots of the mRNA from varioustissues using at least part of the EST clone as a probe can optionallybe performed to check the size of the mRNA against that of the purportedfull length cDNA.

EXAMPLE 5

[0189] Expression via Gene Therapy

[0190] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0191] pMV-7 (Kirschmeier et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith-EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

[0192] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer containing an EcoRI site and the3′ primer further includes a HindIII site. Equal quantities of theMoloney murine sarcoma virus linear backbone and the amplified EcoRI andHindIII fragment are added together, in the presence of T4 DNA ligase.The resulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

[0193] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagle'sMedium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. TheMSV vector containing the gene is then added to the media and thepackaging cells are transduced with the vector. The packaging cells nowproduce infectious viral particles containing the gene (the packagingcells are now referred to as producer cells).

[0194] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0195] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

[0196] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described. TABLE 1 PUTATIVE CLONE NO.IDENTIFICATION SEQ ID NO. HBGBA67 amyloid-like protein 1 present inbreast HE2CB95 hADA2 2 HTEAZ96 TRF 3 HPTIK55 hRPB11 4 HARA063 IRF3 5HLTAH80 TM4SF 6 HNFBT92 TNFR AF1, C1 7 HTPBA27 TM4SF, CD53 8 HLHAR55Retinoid X Receptor 9 HSRDG78 RBP-26 10 HCCAA03 Protein kinase C 11inhibitor protein

[0197] TABLE 2 PUTATIVE CLONE NO. IDENTIFICATION SEQ ID NO. HBGBA67amyloid-like protein 12 present in breast HE2CB95 hADA2 13 HTEAZ96 TRF14 HPTIK55 hRPB11 15 HARA063 IRF3 16 HLTAH80 TM4SF 17 HNFBT92 TNFR AF1,C1 18 HTPBA27 TM4SF, CD53 19 HLHAR55 Retinoid X Receptor 20 HSRDG78RBP-26 21 HCCAA03 Protein kinase C 22 inhibitor protein

[0198]

1 22 550 base pairs nucleic acid single linear DNA 1 CACGAGCCAC C ATGGAT GTT TTC AAG AAG GGC TTC TCC ATC GCC AAG AAG 50 Met Asp Val Phe LysLys Gly Phe Ser Ile Ala Lys Lys 1 5 10 GGC GTG GTG GGT GCG GTG GAA AAGACC AAG CAG GGG GTG ACG GAA GCA 98 Gly Val Val Gly Ala Val Glu Lys ThrLys Gln Gly Val Thr Glu Ala 15 20 25 GCT GAG AAG ACC AAG GAG GGG GTC ATGTAT GTG GGA GCC AAG ACC AAG 146 Ala Glu Lys Thr Lys Glu Gly Val Met TyrVal Gly Ala Lys Thr Lys 30 35 40 45 GAG AAT GTT GTA CAG AGC GTG ACC TCAGTG GCC GAG AAG ACC AAG GAG 194 Glu Asn Val Val Gln Ser Val Thr Ser ValAla Glu Lys Thr Lys Glu 50 55 60 CAG GCC AAC GCC GTG AGC AAG GCT GTG GTGAGC AGC GTC AAC ACT GTG 242 Gln Ala Asn Ala Val Ser Lys Ala Val Val SerSer Val Asn Thr Val 65 70 75 GCC ACC AAG ACC GTG GAG GAG GCG GAG AAC ATCGCG GTC ACC TCC GGG 290 Ala Thr Lys Thr Val Glu Glu Ala Glu Asn Ile AlaVal Thr Ser Gly 80 85 90 GTG GTG CGC AAG GAG GAC TTG AGG CCA TCT GCC CCCCAA CAG GAG GGT 338 Val Val Arg Lys Glu Asp Leu Arg Pro Ser Ala Pro GlnGln Glu Gly 95 100 105 GAG GCA TCC AAA GAG AAA GAG GAA GTG GCA GAG GAGGCC CAG AGT GGG 386 Glu Ala Ser Lys Glu Lys Glu Glu Val Ala Glu Glu AlaGln Ser Gly 110 115 120 125 GGA GAC T AGAGGGCTAC AGGCCAGCGT GGATGACCTGAAGAGCGCTC CTCTGCCTTG 443 Gly Asp GACACCATCC CCTCCTAGCA CAAGGAGTGCCCGCCTTGAG TGACATGCGG GTGCCCACGC 503 TCCTGCCCTC GTCTCCCTGG ACACCCTTGGCCTGTCCACC TGTGCTG 550 1720 base pairs nucleic acid single linear DNA 2AATTCCTGGG GGGTCTCGGC GAGGGAGTCA TCAAGCTTTG GTGTATGTGT TGGCCGGTTC 60TGAAGTCTTG AAGAAGCTCT GCTGAGGAAG ACCAAAGCAG CACTCGTTGC CAATTAGGGA 120ATG GAC CGT TTG GGT TCC TTT AGC AAT GAT CCC TCT GAT AAG CCA CCT 168 MetAsp Arg Leu Gly Ser Phe Ser Asn Asp Pro Ser Asp Lys Pro Pro 5 10 15 TGCCGA GGC TGC TCC TCC TAC CTC ATG GAG CCT TAT ATC AAG TGT GCT 216 Cys ArgGly Cys Ser Ser Tyr Leu Met Glu Pro Tyr Ile Lys Cys Ala 20 25 30 GAA TGTGGG CCA CCT CCT TTT TTC CTC TGC TTG CAG TGT TTC ACT CGA 264 Glu Cys GlyPro Pro Pro Phe Phe Leu Cys Leu Gln Cys Phe Thr Arg 35 40 45 GGC TTT GAGTAC AAG AAA CAT CAA AGC GAT CAT ACT TAT GAA ATA ATG 312 Gly Phe Glu TyrLys Lys His Gln Ser Asp His Thr Tyr Glu Ile Met 50 55 60 ACT TCA GAT TTTCCT GTC CTT GAT CCC AGC TGG ACT GCT CAA GAA GAA 360 Thr Ser Asp Phe ProVal Leu Asp Pro Ser Trp Thr Ala Gln Glu Glu 65 70 75 80 ATG GCC CTT TTAGAA GCT GTG ATG GAC TGT GGC TTT GGA AAT TGG CAG 408 Met Ala Leu Leu GluAla Val Met Asp Cys Gly Phe Gly Asn Trp Gln 85 90 95 GAT GTA GCC AAT CAAATG TGC ACC AAG ACC AAG GAG GAG TGT GAG AAG 456 Asp Val Ala Asn Gln MetCys Thr Lys Thr Lys Glu Glu Cys Glu Lys 100 105 110 CAC TAT ATG AAG CATTTC ATC AAT AAC CCT CTG TTT GCA TCT ACC CTG 504 His Tyr Met Lys His PheIle Asn Asn Pro Leu Phe Ala Ser Thr Leu 115 120 125 CTG AAC CTG AAA CAAGCA GAG GAA GCA AAA ACT GCT GAC ACA GCC ATT 552 Leu Asn Leu Lys Gln AlaGlu Glu Ala Lys Thr Ala Asp Thr Ala Ile 130 135 140 CCA TTT CAC TCT ACAGAT GAC CCT CCC CGA CCT ACC TTT GAC TCC TTG 600 Pro Phe His Ser Thr AspAsp Pro Pro Arg Pro Thr Phe Asp Ser Leu 145 150 155 160 CTT TCT CGG GACATG GCC GGG TAC ATG CCA GCT CGA GCA GAT TTC ATT 648 Leu Ser Arg Asp MetAla Gly Tyr Met Pro Ala Arg Ala Asp Phe Ile 165 170 175 GAG GAA TTT GACAAT TAT GCA GAA TGG GAC TTG AGA GAC ATT GAT TTT 696 Glu Glu Phe Asp AsnTyr Ala Glu Trp Asp Leu Arg Asp Ile Asp Phe 180 185 190 GTT GAA GAT GACTCG GAC ATT TTA CAT GCT CTG AAG ATG GCT GTG GTA 744 Val Glu Asp Asp SerAsp Ile Leu His Ala Leu Lys Met Ala Val Val 195 200 205 GAT ATC TAT CATTCC AGG TTA AAG GAG AGA CAA AGA CGA AAA AAA ATT 792 Asp Ile Tyr His SerArg Leu Lys Glu Arg Gln Arg Arg Lys Lys Ile 210 215 220 ATA AGA GAC CATGGA TTA ATC AAC CTT AGA AAG TTT CAA TTA ATG GAA 840 Ile Arg Asp His GlyLeu Ile Asn Leu Arg Lys Phe Gln Leu Met Glu 225 230 235 240 CGG CGG TATCCC AAG GAG GTC CAG GAC CTG TAT GAA ACA ATG AGG CGA 888 Arg Arg Tyr ProLys Glu Val Gln Asp Leu Tyr Glu Thr Met Arg Arg 245 250 255 TTT GCA AGAATT GTG GGG CCA GTG GAA CAT GAC AAA TTC ATT GAA AGC 936 Phe Ala Arg IleVal Gly Pro Val Glu His Asp Lys Phe Ile Glu Ser 260 265 270 CAT GCA TTGGAA TTT GAA CTC CGA AGG GAA ATC AAG AGG CTC CAA GAA 984 His Ala Leu GluPhe Glu Leu Arg Arg Glu Ile Lys Arg Leu Gln Glu 275 280 285 TAC AGG ACAGCA GGC ATT ACC AAT TTT TGT AGT GCC AGA ACC TAC GAT 1032 Tyr Arg Thr AlaGly Ile Thr Asn Phe Cys Ser Ala Arg Thr Tyr Asp 290 295 300 CAC CTC AAGAAG ACA CGG GAG GAA GAG CGC CTT AAA CGC ACT ATG CTC 1080 His Leu Lys LysThr Arg Glu Glu Glu Arg Leu Lys Arg Thr Met Leu 305 310 315 320 TCA GAAGTT CTC CAG TAT ATC CAG GAC AGT AGT GCT TGC CAG CAG TGG 1128 Ser Glu ValLeu Gln Tyr Ile Gln Asp Ser Ser Ala Cys Gln Gln Trp 325 330 335 CTC CGCCGG CAA GCT GAC ATT GAT TCC GGC CTG AGT CCT TCC ATT CCA 1176 Leu Arg ArgGln Ala Asp Ile Asp Ser Gly Leu Ser Pro Ser Ile Pro 340 345 350 ATG GCTTCG AAT TCA GGT AGA CGG AGT GCA CCA CCC TTG AAC CTC ACT 1224 Met Ala SerAsn Ser Gly Arg Arg Ser Ala Pro Pro Leu Asn Leu Thr 355 360 365 GGC CTCCCT GGC ACA GAG AAG CTG AAT GAA AAA GAA AAG GAG CTC TGT 1272 Gly Leu ProGly Thr Glu Lys Leu Asn Glu Lys Glu Lys Glu Leu Cys 370 375 380 CAG ATGGTG AGG TTG GTC CCT GGA GCC TAT TTA GAA TAC AAA TCT GCT 1320 Gln Met ValArg Leu Val Pro Gly Ala Tyr Leu Glu Tyr Lys Ser Ala 385 390 395 400 CTATTG AAC GAA TGT AAC AAG CAA GGA GGC TTA AGA CTG GCG CAG GCA 1368 Leu LeuAsn Glu Cys Asn Lys Gln Gly Gly Leu Arg Leu Ala Gln Ala 405 410 415 AGAGCA CTC ATC AAG ATA GAT GTG AAC AAA ACC CGG AAA ATC TAT GAT 1416 Arg AlaLeu Ile Lys Ile Asp Val Asn Lys Thr Arg Lys Ile Tyr Asp 420 425 430 TTCCTC ATC AGA GAA GGA TAC ATC ACT AAA GGC T AAGGCTCCAA 1460 Phe Leu IleArg Glu Gly Tyr Ile Thr Lys Gly 435 440 GAGCTTGGGA TCAGAAGTCA GAAGTTTGGAATGTGGTGGG TCAAAGGACA ATATGGGTGG 1520 GCATTCTGGA GAGTTTGTTT TTCAGCTGAATTCTCATGGT GAAAACAGGG GAAAGGACAA 1580 AGGAAACCTT AAGTTGTATT GTCTACTTTCTTCTCCATCC TGCTTTAAAA CACTCCTGTT 1640 GTTGGTATTA TGCTGCAGAG TTGTGTGCTACATAAGCTAT TATTAAATGT GAGTGGGCAT 1700 TCAAAAAAAA AAAAAAAAAA 1720 1537base pairs nucleic acid single linear DNA 3 GGCACGAGGC GCCCCCGGGGTCCCCCGGCC CCTGCAGGGC TACTTGGGCG CAGAGCCGCG 60 GAGGGTCTCC GTTCCTAGAGGTCCTCCTAT CCCGGGCTGC CTGAGTCCTC GCCAGCATCC 120 GCCCTCTCCC ACTCCCATCCTTCCTGGATC CGCCTCTCGG TTCCCGAGGG ACAGTCCCGA 180 CCGCAAACCC ACGTAGAGTAAGGAATGTGG GACAGGCGAC AGAAGTGGCA TACGGTCCTG 240 CGTTATCCCT CCGTCTCGCCACACCTTGTG TCTCCATCTC TCCCCACTTC CTTCCCTCCG 300 TCTGTCATCT GTCATCCCCGGTCGCTCTAA GACCAGGATT CCAATTCGCC TAGTGAGGAA 360 TCTCACTAGG GGAATTTATCGCGACATCAT AAATTAACGG GTTCATTTTG ACTGAAAAGC 420 GAAGGACTTT TTTCAGGCAGAAAACAAGTC TCGTCTGGAC GGATGTGATC TTCGTGGTGG 480 AAAGCTAAAT TTTAAAACCACCCCA ATG GAT GCA GAC AGT GAT GTT GCA TTG 532 Met Asp Ala Asp Ser AspVal Ala Leu 1 5 GAC ATT CTA ATT ACA AAT GTA GTC TGT GTT TTT AGA ACA AGATGT CAT 580 Asp Ile Leu Ile Thr Asn Val Val Cys Val Phe Arg Thr Arg CysHis 10 15 20 25 TTA AAC TTA AGG AAG ATT GCT TTG GAA GGA GCA AAT GTA ATTTAT AAA 628 Leu Asn Leu Arg Lys Ile Ala Leu Glu Gly Ala Asn Val Ile TyrLys 30 35 40 CGT GAT GTT GGA AAA GTA TTA ATG AAG CTT AGA AAA CCT AGA ATTACA 676 Arg Asp Val Gly Lys Val Leu Met Lys Leu Arg Lys Pro Arg Ile Thr45 50 55 GCT ACA ATT TGG TCC TCA GGA AAA ATT ATT TGC ACT GGA GCA ACA AGT724 Ala Thr Ile Trp Ser Ser Gly Lys Ile Ile Cys Thr Gly Ala Thr Ser 6065 70 GAA GAA GAA GCT AAA TTT GGT GCC AGA CGC TTA GCC CGT AGT CTG CAG772 Glu Glu Glu Ala Lys Phe Gly Ala Arg Arg Leu Ala Arg Ser Leu Gln 7580 85 AAA CTA GGT TTT CAG GTA ATA TTT ACA GAT TTT AAG GTT GTT AAC GTT820 Lys Leu Gly Phe Gln Val Ile Phe Thr Asp Phe Lys Val Val Asn Val 9095 100 105 CTG GCA GTG TGT AAC ATG CCA TTT GAA ATC CGT TTG CCA GAA TTCACA 868 Leu Ala Val Cys Asn Met Pro Phe Glu Ile Arg Leu Pro Glu Phe Thr110 115 120 AAG AAC AAT AGA CCT CAT GCC AGT TAC GAA CCT GAA CTT CAT CCTGCT 916 Lys Asn Asn Arg Pro His Ala Ser Tyr Glu Pro Glu Leu His Pro Ala125 130 135 GTG TGC TAT CGG ATA AAA TCT CTA AGA GCT ACA TTA CAG ATT TTTTCA 964 Val Cys Tyr Arg Ile Lys Ser Leu Arg Ala Thr Leu Gln Ile Phe Ser140 145 150 ACA GGA AGT ATC ACA GTA ACA GGG CCC AAT GTA AAG GCT GTT GCTACT 1012 Thr Gly Ser Ile Thr Val Thr Gly Pro Asn Val Lys Ala Val Ala Thr155 160 165 GCT GTG GAA CAG ATT TAC CCA TTT GTG TTT GAA AGC AGG AAA GAAATT 1060 Ala Val Glu Gln Ile Tyr Pro Phe Val Phe Glu Ser Arg Lys Glu Ile170 175 180 185 TTA T AATTCACCAC TTAATTGGTT AGAATCTCTA ACTGAGCACCTTTTAAACCT 1114 Leu GCTGCACATT GGACTCAAAA GGAAAACTGG ACCAACAATAATTGAGGAAA TAGACTTTTT 1174 TATTCATTCA CGGCTACAGT GTAAGCTCCA GTCCCTTTGGATTTTATTCC AAACCTTGCT 1234 GTAATATAAA AGGAAGTTTA CAAGACATGA TATTGCTGCTTTTACAAAAG GACATTCTAT 1294 TTATTTTCGC AGTAATTCTC ATGTCCCCAT AAGCAGAGCTGTCACAGTGT GCACTACCTT 1354 AGATTGTTTT ATTGTCGTCA TTGTTATTTT TTTCCATTTGGAGCTAATGT GTTTTATTTG 1414 TGAATAGTCT TTTACATTTT TGTATGCTGA ATATGGGCACCAAAGAACCT GTAAAAGTTA 1474 TCTTTTTCAA TTGAATGTGC ACAAATAAAA GTTTGGAAAGAAAAAAAAAA AAAAAAAAAA 1534 AAA 1537 555 base pairs nucleic acid singlelinear DNA 4 ACGAGCAACG GCGGCGGGAG C ATG AAC GCC CCT CCA GCC TTC GAG TCGTTC 51 Met Asn Ala Pro Pro Ala Phe Glu Ser Phe 1 5 10 TTG CTC TTC GAGGGC GAG AAG AAG ATC ACC ATT AAC AAG GAC ACC AAG 99 Leu Leu Phe Glu GlyGlu Lys Lys Ile Thr Ile Asn Lys Asp Thr Lys 15 20 25 GTA CCC AAT GCC TGTTTA TTC ACC ATC AAC AAA GAA GAC CAC ACA CTG 147 Val Pro Asn Ala Cys LeuPhe Thr Ile Asn Lys Glu Asp His Thr Leu 30 35 40 GGA AAC ATC ATT AAA TCACAA CTC CTA AAA GAC CCG CAA GTG CTA TTT 195 Gly Asn Ile Ile Lys Ser GlnLeu Leu Lys Asp Pro Gln Val Leu Phe 45 50 55 GCT GGC TAC AAA GTC CCC CACCCC TTG GAG CAC AAG ATC ATC ATC CGA 243 Ala Gly Tyr Lys Val Pro His ProLeu Glu His Lys Ile Ile Ile Arg 60 65 70 GTG CAG ACC ACG CCG GAC TAC AGCCCC CAG GAA GCC TTT ACC AAC GCC 291 Val Gln Thr Thr Pro Asp Tyr Ser ProGln Glu Ala Phe Thr Asn Ala 75 80 85 90 ATC ACC GAC CTC ATC AGT GAG CTGTCC CTG CTG GAG GAG CGC TTT CGG 339 Ile Thr Asp Leu Ile Ser Glu Leu SerLeu Leu Glu Glu Arg Phe Arg 95 100 105 GTG GCC ATA AAA GAC AAG CAG GAAGGA ATT GAG T AGGGGCCAGA 383 Val Ala Ile Lys Asp Lys Gln Glu Gly Ile Glu110 115 GGGGGCTCTG CTCGGCCTGT GAGCCCCGTT CCTACCTGTG CCTGACCCTCCGCTCCAGGT 443 ACCACACCGA GGAGAGCGGC CAGTCCCAGC CATGGCCCGC CTTGTGGCCACCCCTCACCC 503 TGACACCGAC GTGTCCTGTA CATAGATTAG GTTTTATATT CCTAATAAAG TA555 1426 base pairs nucleic acid single linear DNA 5 GGTTCCAGCTGCCCGCACGC CCCGACCTTC CATCGTAGGC CGGACCATGG GAACCCCAAA 60 GCCACGGNTCCTGCCCTGGC TGGTGTCGCA GCTGGACCTG GGGCAACTGG AGGGCGTGGC 120 CTGGGTGAACAAGAGCCGCA CGCGCTTCCG CATCCCTTGG AAGCACGGCC TACGGCAGGA 180 TGCACAGCAGGAGGATTTCG GAATCTTCCA GGCCTGGGCC GAGGCCACTG GTGCATATGT 240 TCCCGGGAGGGATAAGCCAG ACCTGCCAAC CTGGAAGAGG AATTTCCGCT CTGCCCTCAA 300 CCGCAAAGAAGGGTTGCGTT TAGCAGAGGA CCGGAGCAAG GACCCTCACG ACCCACATAA 360 AATCTACGAGTTTGTGAACT CAGGAGTTGG GGACTTTTCC CAGCCAGACA CCTCTCCGGA 420 CACCAATGGTGGAGGCAGTA CTTCTGATAC CCAGGAAGAC ATTCTGGATG AGTTACTGGG 480 TAACATGGTGTTGGCCCCAC TCCCAGATCC GGGACCCCCA AGCCTGGCTG TAGCCCCTGA 540 GCCCTGCCCTCAGCCCCTGC GGAGCCCCAG CTTGGACAAT CCCACTCCCT TCCCAAACCT 600 GGGGCCCTCTGAGAACCCAC TGAAGCGGCT GTTGGTGCCG GGGGAAGAGT GGGAGTTCGA 660 GGTGACAGCCTTCTACCGGG GCCGCCAAGT CTTCCAGCAG ACCATCTCCT GCCCGGAGGG 720 CCTGCGGCTGGTGGGGTCCG AAGTGGGAGA CAGGACGCTG CCTGGATGGC CAGTCACACT 780 GCCAGACCCTGGCATGTCCC TGACAGACAG GGGAGTGATG AGCTACGTGA GGCATGTGCT 840 GAGCTGCCTGGGTGGGGGAC TGGCTCTCTG GCGGGCCGGG CAGTGGCTCT GGGCCCAGCG 900 GCTGGGGCACTGCCACACAT ACTGGGCAGT GAGCGAGGAG CTGCTCCCCA ACAGCGGGCA 960 TGGGCCTGATGGCGAGGTCC CCAAGGACAA GGAAGGAGGC GTGTTTGACC TGGGGCCCTT 1020 CATTGTAGATCTGATTACCT TCACGGAAGG AAGCGGACGC TCACCACGCT ATGCCCTCTG 1080 GTTCTGTGTGGGGGAGTCAT GGCCCCAGGA CCAGCCGTGG ACCAAGAGGC TCGTGATGGT 1140 CAAGGTTGTGCCCACGTGCC TCAGGGCCTT GGTAGAAATG GCCCGGGTAG GGGGTGCCTC 1200 CTCCCTGGAGAATACTGTGG ACCTGCACAT TTCCAACAGC CACCCACTCT CCCTCACCTC 1260 CGACCAGTACAAGGCCTACC TGCAGGACTT GGTGGAGGGC ATGGATTTCC AGGGCCCTGG 1320 GGAGAGCTGAGCCCTCGCTC CTCATGGTGT GCCTCCAACC CCCCTGTTCC CCACCACCTC 1380 AACCAATAAACTGGTTCCTG CTATGAAAAA AAAAAAAAAA AAAAAA 1426 1001 base pairs nucleicacid single linear DNA 6 AATTCGGCAG AGGCAGTTCC TAGCGAGGAG GCGCCGCGCATTGCCGCTCT CTCGGTGAGC 60 GCAGCCCGCT CTCCGGGCCG GGCCTTCGCG GGCCACCGCG CCATG GGC CAG TGC 114 Met Gly Gln Cys 1 GGC ATC ACC TCC TCC AAG ACC GTGCTG GTC TTT CTC AAC CTC ATC TTC 162 Gly Ile Thr Ser Ser Lys Thr Val LeuVal Phe Leu Asn Leu Ile Phe 5 10 15 20 TGG GGG GCA GCT GGC ATT TTA TGCTAT GTG GGA GCC TAT GTC TTC ATC 210 Trp Gly Ala Ala Gly Ile Leu Cys TyrVal Gly Ala Tyr Val Phe Ile 25 30 35 ACT TAT GAT GAC TAT GAC CAC TTC TTTGAA GAT GTG TAC ACG CTC ATC 258 Thr Tyr Asp Asp Tyr Asp His Phe Phe GluAsp Val Tyr Thr Leu Ile 40 45 50 CCT GCT GTA GTG ATC ATA GCT GTA GGA GCCCTG CTT TTC ATC ATT GGG 306 Pro Ala Val Val Ile Ile Ala Val Gly Ala LeuLeu Phe Ile Ile Gly 55 60 65 CTA ATT GGC TGC TGT GCC ACA ATC CGG GAA AGTCGC TGT GGA CTT GCC 354 Leu Ile Gly Cys Cys Ala Thr Ile Arg Glu Ser ArgCys Gly Leu Ala 70 75 80 ACG TTT GTC ATC ATC CTG CTC TTG GTT TTT GTC ACAGAA GTT GTT GTA 402 Thr Phe Val Ile Ile Leu Leu Leu Val Phe Val Thr GluVal Val Val 85 90 95 100 GTG GTT TTG GGA TAT GTT TAC AGA GCA AAG GTG GAAAAT GAG GTT GAT 450 Val Val Leu Gly Tyr Val Tyr Arg Ala Lys Val Glu AsnGlu Val Asp 105 110 115 CGC AGC ATT CAG AAA GTG TAT AAG ACC TAC AAT GGAACC AAC CCT GAT 498 Arg Ser Ile Gln Lys Val Tyr Lys Thr Tyr Asn Gly ThrAsn Pro Asp 120 125 130 GCT GCT AGC CGG GCT ATT GAT TAT GTA CAG AGA CAGCTG CAT TGT TGT 546 Ala Ala Ser Arg Ala Ile Asp Tyr Val Gln Arg Gln LeuHis Cys Cys 135 140 145 GGA ATT CAC AAC TAC TCA GAC TGG GAA AAT ACA GATTGG TTC AAA GAA 594 Gly Ile His Asn Tyr Ser Asp Trp Glu Asn Thr Asp TrpPhe Lys Glu 150 155 160 ACC AAA AAC CAG AGT GTC CCT CTT AGC TGC TGC AGAGAG ACT GCC AGC 642 Thr Lys Asn Gln Ser Val Pro Leu Ser Cys Cys Arg GluThr Ala Ser 165 170 175 180 AAT TGT AAT GGC AGC TGG CCA CCC TTC CGA CTCTAT GCT GAG GGG TGT 690 Asn Cys Asn Gly Ser Trp Pro Pro Phe Arg Leu TyrAla Glu Gly Cys 185 190 195 GAG GCT CTA GTT GTG AAG AAG CTA CAA GAA ATCATG ATG CAT GTG ATC 738 Glu Ala Leu Val Val Lys Lys Leu Gln Glu Ile MetMet His Val Ile 200 205 210 TGG GCC GCA CTG GCA TTT GCA GCT ATT CAG CTGCTG GGC ATG CTG TGT 786 Trp Ala Ala Leu Ala Phe Ala Ala Ile Gln Leu LeuGly Met Leu Cys 215 220 225 GCT TGC ATC GTG TTG TGC AGA AGG AGT AGA GATCCT GCT TAC GAG CTC 834 Ala Cys Ile Val Leu Cys Arg Arg Ser Arg Asp ProAla Tyr Glu Leu 230 235 240 CTC ATC ACT GGC GGA ACC TAT GCA TAG TTGACAACTCA AGCCTGAGCT 882 Leu Ile Thr Gly Gly Thr Tyr Ala 245 250TTTTGGTCTT GTTCTGATTT GGAAGGTGAA TTGAGCAGGT CTGCTGCTGT TGGCCTCTGG 942AGTTCATCTA GTTAAAGCAC ATGTACACTG GTGTTGGACA GAGCAGCTTG GCTTTTCAT 10012361 base pairs nucleic acid single linear DNA 7 GAGCCAGGAC TCCACAAGGCTGGTCCCCTG CCCTGGAGCA ACTTAAACAG GCCCTCTGGC 60 CAGCCTGGAA CCCTGAG ATGGCC TCC AGC TCA GGC AGC AGT CCT CGC CCG 110 Met Ala Ser Ser Ser Gly SerSer Pro Arg Pro 1 5 10 GCC CCT GAT GAG AAT GAG TTT CCC TTT GGG TGC CCTCCC ACC GTC TGC 158 Ala Pro Asp Glu Asn Glu Phe Pro Phe Gly Cys Pro ProThr Val Cys 15 20 25 CAG GAC CCA AAG GAG CCC AGG GCT CTC TGC TGT GCA GGCTGT CTC TCT 206 Gln Asp Pro Lys Glu Pro Arg Ala Leu Cys Cys Ala Gly CysLeu Ser 30 35 40 GAG AAC CCG AGG AAT GGC GAG GAT CAG ATC TGC CCC AAA TGCAGA GGG 254 Glu Asn Pro Arg Asn Gly Glu Asp Gln Ile Cys Pro Lys Cys ArgGly 45 50 55 GAA GAC CTC CAG TCT ATA AGC CCA GGA AGC CGT CTT CGA ACT CAGGAG 302 Glu Asp Leu Gln Ser Ile Ser Pro Gly Ser Arg Leu Arg Thr Gln Glu60 65 70 75 AAG GTT CAG GCG GAG GTC GCT GAG GCT GGG ATT GGG TGC CCC TTTGCT 350 Lys Val Gln Ala Glu Val Ala Glu Ala Gly Ile Gly Cys Pro Phe Ala80 85 90 GTT GTC GGC TGC TCC TTC AAG GGA AGC CCA CAG TTT GTG GAA GAG CAT398 Val Val Gly Cys Ser Phe Lys Gly Ser Pro Gln Phe Val Glu Glu His 95100 105 GAG GTC ACC TCC CAG ACC TCC CAC CTA AAC CTG CTG TTG GGG TTC ATG446 Glu Val Thr Ser Gln Thr Ser His Leu Asn Leu Leu Leu Gly Phe Met 110115 120 AAA CAG TGG AAG GCC CGG CTG GGC TGT GGC CTG GAT TCT GGG CCC ATG494 Lys Gln Trp Lys Ala Arg Leu Gly Cys Gly Leu Asp Ser Gly Pro Met 125130 135 GCC CTG GAG CAG AAC CTG TCA GAC CTG CAG CTG CAG GCA GCC GTG GAA542 Ala Leu Glu Gln Asn Leu Ser Asp Leu Gln Leu Gln Ala Ala Val Glu 140145 150 155 GTG GCG GGG GAC CTG GAG GTC GAT TGC TAC CGG GCA CCC TGC TCCGAG 590 Val Ala Gly Asp Leu Glu Val Asp Cys Tyr Arg Ala Pro Cys Ser Glu160 165 170 AGC CAG GAG GAG CTG GCC CTG CAG CAC TTC ATG AAG GAG AAG CTTCTG 638 Ser Gln Glu Glu Leu Ala Leu Gln His Phe Met Lys Glu Lys Leu Leu175 180 185 GCT GAG CTG GAG GGG AAG CTG CGT GTG TTT GAG AAC AAT GTT GCTGTC 686 Ala Glu Leu Glu Gly Lys Leu Arg Val Phe Glu Asn Asn Val Ala Val190 195 200 CTC AAC AAG GAG GTG GAG GCC TCC CAC CTG GCC CTG GCC ACC TCTATC 734 Leu Asn Lys Glu Val Glu Ala Ser His Leu Ala Leu Ala Thr Ser Ile205 210 215 CAC CAG AGC CAG CTG GAC CGT GAG CGC ATC CTG AGC TTG GAG CAGAGG 782 His Gln Ser Gln Leu Asp Arg Glu Arg Ile Leu Ser Leu Glu Gln Arg220 225 230 235 GTG GTG CAG GTT CAG CAG ACC CTG GCC CAG AAA GAC CAG GCCCTG GGC 830 Val Val Gln Val Gln Gln Thr Leu Ala Gln Lys Asp Gln Ala LeuGly 240 245 250 AAG CTG GAG CAG AGC TTG CGC CTC ATG GAG GAG GCC TCC TTCGAT GGC 878 Lys Leu Glu Gln Ser Leu Arg Leu Met Glu Glu Ala Ser Phe AspGly 255 260 265 ACT TTC CTG TGG AAG ATC ACC AGT GTC ACC AGG CGG TGC CATGAG TCG 926 Thr Phe Leu Trp Lys Ile Thr Ser Val Thr Arg Arg Cys His GluSer 270 275 280 GCC TGT GGC AGG ACC GTC AGC CTC TTC TCC CCA GCC TTC TACACT GCC 974 Ala Cys Gly Arg Thr Val Ser Leu Phe Ser Pro Ala Phe Tyr ThrAla 285 290 295 AAG TAT GGC TAC AAG TTG TGC CTG CGG CTG TAC CTG ATT GGAGAT GGC 1022 Lys Tyr Gly Tyr Lys Leu Cys Leu Arg Leu Tyr Leu Ile Gly AspGly 300 305 310 315 ACT GGA AAG AGA ACC CAT CTT TCG CTC TTC ATC GTG ATCATG AGA GGG 1070 Thr Gly Lys Arg Thr His Leu Ser Leu Phe Ile Val Ile MetArg Gly 320 325 330 GAG TAT GAT GCG CTG CTG CCG TGG CCT TTC CGG AAC AAGGTC ACC TTC 1118 Glu Tyr Asp Ala Leu Leu Pro Trp Pro Phe Arg Asn Lys ValThr Phe 335 340 345 ATG CTG CTG GAC CAG AAC AAC CGT GAG CAC GCC ATT GACGCC TTC CGG 1166 Met Leu Leu Asp Gln Asn Asn Arg Glu His Ala Ile Asp AlaPhe Arg 350 355 360 CCT GAC CTA AGC TCA GCG TCC TTC CAG AGG CCC CAG AGTGAA ACC AAC 1214 Pro Asp Leu Ser Ser Ala Ser Phe Gln Arg Pro Gln Ser GluThr Asn 365 370 375 GTG GCC AGT GGA TGC CCA CTC TTC TTC CCC CTC AGC AAACTG CAG TCA 1262 Val Ala Ser Gly Cys Pro Leu Phe Phe Pro Leu Ser Lys LeuGln Ser 380 385 390 395 CCC AAG CAC GCC TAC GTA AGG ACG ACA CAA TGT TCCTCA AGT GCA TTG 1310 Pro Lys His Ala Tyr Val Arg Thr Thr Gln Cys Ser SerSer Ala Leu 400 405 410 TGG AGA CCA GCA CTT AGG GTGGGCGGGG CTCCTGAGGGAGTTCCAACT 1358 Trp Arg Pro Ala Leu Arg 415 CAGAAGGGAG CTAGCCAGAGGACTGTGATG CCCTGCCCTT GGCACCCAAG AACTCAGGGC 1418 ACAAAGATGG GTGAAGGCTGGCATGATCCA AGCAAGATGA GGGGTCGATT CGGGTGGCCA 1478 TCTGGTTAGA TGGCAGGACGTGGGTGGGCC CACAAAGGCA AAGGGTCCAG AAGGAGACAG 1538 GCAGAGCTGC TCCCCTCTGCACGGACCATG CGACACTGGG AGGCCAGTGA GCCACTCCGG 1598 CCCCGAATGT TGAGGTGGACTCTCACCAAA TGAGAAGAAA ATGGAACCAG GCTTGGAACC 1658 GTAGGACCCA AGCAGAGAAGCTCTCGGGCT AGGAAGATCT CTGCAGGGCC GCCAGGGAGA 1718 CCTGGACACA GGCCTGCTCTCTTTTTCTCC AGGGTCAGAA ACAGGACCGG GTGGAAGGGA 1778 TGGGGTGCCA GTTTGAATGCAGTCTGTCCA GGCTCGTCAT TGGAGGTGAA CAAGCAAACC 1838 CAGACGGCTC CACTAGGACTTCAAATTGGG GGTTGGATTT GAAGACTTTT AAGTTTCCTT 1898 CCAGCCCAGA AAGTCTCTCATTCTAGCCTC CTGGCCCAGG TGAGTCCTAG AGCTACAGGG 1958 GTTCTGGAAA CATTCAGGAGCTTCCTGTCC TCCCAGCTCC TCACTCACCT TCAGTAACCC 2018 CCACTGGACT GACCTGGTCCACAGGGCACC TGCCACCCTG GGCCTGGCAG CTCAGCTTCC 2078 CAACACGCAG GAGCACACCCAGCCCCCACA TCCTGTGCCT CCATCAGCTA AACACCACGT 2138 CACTTCATGC AGGTGAAACCCAGTCACTGT GAGCTCCCAG GTGCAGCCAG AGGCACCTCA 2198 AGAAGAAGAG GGGCATAAACTTTCCTCTTC CTGCCTAGAG GCCCCACCTT TGGTGCTTTC 2258 CAGAATCCCG TAACACCTGATTAACTGAGG CATCCACTTC TTTCAGCAGA CTGATCAGGA 2318 CCTCCAAGCC ACTGAGCAATGTATAACCCC AAAGGGAATT CAA 2361 1344 base pairs nucleic acid singlelinear DNA 8 GGCGAGGCTG CTGGTGGCTG TGGAGAGCTT GGGGCTTCCT TGGTCGCACCCACCACCTGC 60 CTGCCCACTG GTCAGCCTTC AGGGAACCCT GAGCACCGCC TGGTCTCTTTCCTGTGGCCA 120 GCCCAGAACT GAAGCGCTGC GGC ATG GCG CGC GCC TGC CTC CAG GCCGTC 170 Met Ala Arg Ala Cys Leu Gln Ala Val 1 5 AAG TAC CTC ATG TTC GCCTTC AAC CTG CTC TTC TGG CTG GGA GGC TGT 218 Lys Tyr Leu Met Phe Ala PheAsn Leu Leu Phe Trp Leu Gly Gly Cys 10 15 20 25 GGC GTG CTG GGT GTC GGCATC TGG CTG GCC GCA CAA CAG GGG AGC TTT 266 Gly Val Leu Gly Val Gly IleTrp Leu Ala Ala Gln Gln Gly Ser Phe 30 35 40 GCC ACG CTG TCC TCT TCC TTCCCG TCC CTG TGG GCT GCC AAC CTG CTC 314 Ala Thr Leu Ser Ser Ser Phe ProSer Leu Trp Ala Ala Asn Leu Leu 45 50 55 ATC ATC ACC GGC GCC TTT GTC ATGGCC ATC GGC TTC GTG GGC TGC CTG 362 Ile Ile Thr Gly Ala Phe Val Met AlaIle Gly Phe Val Gly Cys Leu 60 65 70 GGT GCC ATC AAG GAG AAC AAG TGC CTCCTG CTC ACT TTC TTC CTG CTG 410 Gly Ala Ile Lys Glu Asn Lys Cys Leu LeuLeu Thr Phe Phe Leu Leu 75 80 85 CTG CTG CTG GTG TTC CTG CTG GAG GGC ACCATC GCC ATC CTC TTC TTC 458 Leu Leu Leu Val Phe Leu Leu Glu Gly Thr IleAla Ile Leu Phe Phe 90 95 100 105 GCC TAC ACG GAC AAG ATT GAC AGG TATGCC CAG CAA GAC CTG AAG AAA 506 Ala Tyr Thr Asp Lys Ile Asp Arg Tyr AlaGln Gln Asp Leu Lys Lys 110 115 120 GGC TTG CAC CTG TAC GGC ACG CAG GGCAAC GTG GGC CTC ACC AAC GCC 554 Gly Leu His Leu Tyr Gly Thr Gln Gly AsnVal Gly Leu Thr Asn Ala 125 130 135 TGG AGC ATC ATC CAG ACC GAC TTC CGCTGC TGT GGC GTC TCC AAC TAC 602 Trp Ser Ile Ile Gln Thr Asp Phe Arg CysCys Gly Val Ser Asn Tyr 140 145 150 ACT GAC TGG TTC GAG GTG TAC AAC GCCACG CGG GTA CCT GAC TCC TGC 650 Thr Asp Trp Phe Glu Val Tyr Asn Ala ThrArg Val Pro Asp Ser Cys 155 160 165 TGC TTG GAG TTC AGT GAG AGC TGT GGGCTG CAC GCC CCG GCA CTG GTG 698 Cys Leu Glu Phe Ser Glu Ser Cys Gly LeuHis Ala Pro Ala Leu Val 170 175 180 185 GAG GGC CGT GCT ACG AGA GGT GAAGGT GTG GCT TCA GGA GAA CTG CTG 746 Glu Gly Arg Ala Thr Arg Gly Glu GlyVal Ala Ser Gly Glu Leu Leu 190 195 200 GCT GTG GGC ATC TTT GGG CTG TGCACG GCG CTG GTG CAG ATC CTG GGC 794 Ala Val Gly Ile Phe Gly Leu Cys ThrAla Leu Val Gln Ile Leu Gly 205 210 215 CTG AAC TTC GCC ATG ACC ATG TACTGG CAA GTG GTC AAG GCA GAC ACC 842 Leu Asn Phe Ala Met Thr Met Tyr TrpGln Val Val Lys Ala Asp Thr 220 225 230 TAC TGT GCG TAG G CCCCCCACCGCCCGCTTCTC TTTCAAAAGG ACGCCCACGG 895 Tyr Cys Ala 235 GGAGATGGCCGCACCCACAG AGTGTCTTTC CCACCACCAG CCTCGGTGCT CTTTCCCATG 955 CTGGGAGGAGGGAGGGAGGG AAAGTTGCCT GGAGCCCCCG GAACCCTGTT TCTGGAAGGC 1015 CCTAGCTCAGGTGGCTTTCA GGGCCTCCGG ACCCCCCCTG GGAAGGGTGG CCACGTGCTG 1075 GCTTCGGAACCCAGGGCAGG GGTGGGAGGG GCCTCCAGCA CTTTTTATAT TTACGTATTC 1135 TCCAAAACAGTGTTCACACG GGAGCCAACC TGTGGCCCCC AGCCTCCTGG AAAAAAGGTT 1195 GGCGCTGGAGGAACCGGGTC TTGGCATCCT GGAGGTGGCC CCACTGGTCC TGGTGCTCCA 1255 GGCGGGGCCGTGGACCCCTC ACCTACATTC CATAGTGGGC CCGTGGGGCT CCTGGTGCAT 1315 CTTAATAAAGTGTGAGCAGC AAAAAAAAA 1344 1641 base pairs nucleic acid single linear DNA9 AGCCCAAGTT GAAGAAAGCC GGGCTGTGCC TGGAAGCCGA GAGAGGCGGT AATATTTAGA 60AGCTGCACAG GAGAGGAACA TGAACTGACG AGTAAAC ATG TAT GGA AAT TAT TCT 115 MetTyr Gly Asn Tyr Ser 1 5 CAC TTC ATG AAG TTT CCC GCA GGC TAT GGA GGC TCCCCT GGC CAC ACT 163 His Phe Met Lys Phe Pro Ala Gly Tyr Gly Gly Ser ProGly His Thr 10 15 20 GGC TCT ACA TCC ATG AGC CCA TCA GCA GCC TTG TCC ACAGGG AAG CCA 211 Gly Ser Thr Ser Met Ser Pro Ser Ala Ala Leu Ser Thr GlyLys Pro 25 30 35 ATG GAC AGC CAC CCC AGC TAC ACA GAT ACC CCA GTG AGT GCCCCA CGG 259 Met Asp Ser His Pro Ser Tyr Thr Asp Thr Pro Val Ser Ala ProArg 40 45 50 ACT CTG AGT GCA GTG GGG ACC CCC CTC AAT GCC CTG GGC TCT CCATAT 307 Thr Leu Ser Ala Val Gly Thr Pro Leu Asn Ala Leu Gly Ser Pro Tyr55 60 65 70 CGA GTC ATC ACC TCT GCC ATG GGC CCA CCC TCA GGA GCA CTT GCAGCG 355 Arg Val Ile Thr Ser Ala Met Gly Pro Pro Ser Gly Ala Leu Ala Ala75 80 85 CCT CCA GGA ATC AAC TTG GTT GCC CCA CCC AGC TCT CAG CTA AAT GTG403 Pro Pro Gly Ile Asn Leu Val Ala Pro Pro Ser Ser Gln Leu Asn Val 9095 100 GTC AAC AGT GTC AGC AGT TCA GAG GAC ATC AAG CCC TTA CCA GGG CTT451 Val Asn Ser Val Ser Ser Ser Glu Asp Ile Lys Pro Leu Pro Gly Leu 105110 115 CCC GGG ATT GGA AAC ATG AAC TAC CCA TCC ACC AGC CCC GGA TCT CTG499 Pro Gly Ile Gly Asn Met Asn Tyr Pro Ser Thr Ser Pro Gly Ser Leu 120125 130 GTT AAA CAC ATC TGT GCT ATC TGT GGA GAC AGA TCC TCA GGA AAG CAC547 Val Lys His Ile Cys Ala Ile Cys Gly Asp Arg Ser Ser Gly Lys His 135140 145 150 TAC GGG GTA TAC AGT TGT GAA GGC TGC AAA GGG TTC TTC AAG AGGACG 595 Tyr Gly Val Tyr Ser Cys Glu Gly Cys Lys Gly Phe Phe Lys Arg Thr155 160 165 ATA AGG AAG GAC CTC ATC TAC ACG TGT CGG GAT AAT AAA GAC TGCCTC 643 Ile Arg Lys Asp Leu Ile Tyr Thr Cys Arg Asp Asn Lys Asp Cys Leu170 175 180 ATT GAC AAG CGT CAG CGC AAC CGC TGC CAG TAC TGT CGC TAT CAGAAG 691 Ile Asp Lys Arg Gln Arg Asn Arg Cys Gln Tyr Cys Arg Tyr Gln Lys185 190 195 TGC CTT GTC ATG GGC ATG AAG AGG GAA GCT TGT GCA AAG AAG GAAAGA 739 Cys Leu Val Met Gly Met Lys Arg Glu Ala Cys Ala Lys Lys Glu Arg200 205 210 CAG AGG AGC CGA GAG CGA GCT GAG AGT GAG GCA GAA TGT GCT ACCAGT 787 Gln Arg Ser Arg Glu Arg Ala Glu Ser Glu Ala Glu Cys Ala Thr Ser215 220 225 230 GGT CAT GAA GAC ATG CCT GTG GAG AGG ATT CTA GAA GCT GAACTT GCT 835 Gly His Glu Asp Met Pro Val Glu Arg Ile Leu Glu Ala Glu LeuAla 235 240 245 GTT GAC CCA AAG ACA GAA TCC TAT GGT GAC ATG AAT ATG GAGAAC TCG 883 Val Asp Pro Lys Thr Glu Ser Tyr Gly Asp Met Asn Met Glu AsnSer 250 255 260 ACA AAT GAC CCT GTT ACC AAC ATA TGT CAT GCT GCT GAC AAGCAG CTT 931 Thr Asn Asp Pro Val Thr Asn Ile Cys His Ala Ala Asp Lys GlnLeu 265 270 275 CAC ACC CTC GGT GAA TGG GCC AAG CGT ATT CCC CAC TTC TCTGAC CTC 979 His Thr Leu Gly Glu Trp Ala Lys Arg Ile Pro His Phe Ser AspLeu 280 285 290 ACC TTG GAG GAC CAG GTC ATT GTG CTT CGG ACA GGG TGG AATGAA TTG 1027 Thr Leu Glu Asp Gln Val Ile Val Leu Arg Thr Gly Trp Asn GluLeu 295 300 305 310 CTG ATT GCC TCT TTC TCC CAC CGC TCA GTT TCC GTG GAGGAT GGC ATC 1075 Leu Ile Ala Ser Phe Ser His Arg Ser Val Ser Val Glu AspGly Ile 315 320 325 CCT CTG GCC ACG GGT TTA CAT GTC CAC CGG AGC AGT GCCCAC AG 1119 Pro Leu Ala Thr Gly Leu His Val His Arg Ser Ser Ala His 330335 340 TGCTGGGGTC GGCTCCATCT TTGACAGAGC TCTAACTGAG CTGGTTTCCAAACTGAAAGA 1179 CATGCAGGTG GACAAGTCGG AACTGGGATG CCTGCGAGCC ATTGTTCTCTTTCAACCCCA 1239 GATGCCCAAG GGCCTGCCCA CCCCCTTTGA GGTGGAGACT CTGCGAAAGAAGGTTTATGC 1299 CACCCTTGAG GCCCACCACC AAGCAGAATA TCCGGAACAG CCAGGCAAGGTTTGCCAAGC 1359 TGCTGTGCGC CTCCCAGCTC TGCGTTCCAT TGGCTTGAAA TGCCTGGAGCACCTCTTCTT 1419 CTTCAAGCTC ATCGGGGACA CCCCCATTGA CACCTTCCTC ATGGAGATGTTGGAGACCCC 1479 GCTGCAGATC ACCTGAGCCC CACCAGCCAA AGCCTCCCCA CCCAGGATGACCCCTGGGCA 1539 GGTGTGTGTG GACCCCCACC CTGCACTTTC CTCCACCTCC CACCCTGACCCCCTTCCTGT 1599 CCCCAAAATG TGATGCTTAT AATAAAGAAA ACCTTTCTAC AA 1641 1185base pairs nucleic acid single linear DNA 10 ATG GCG TTG GAG GTC GGC GATATG GAA GAT GGG CAG CTT TCC GAC TCG 48 Met Ala Leu Glu Val Gly Asp MetGlu Asp Gly Gln Leu Ser Asp Ser 1 5 10 15 GAT TCC GAC ATG ACG GTC GCACCC AGC GAC AGG CCG CTG CAA TTG CCA 96 Asp Ser Asp Met Thr Val Ala ProSer Asp Arg Pro Leu Gln Leu Pro 20 25 30 AAA GTG CTA GGT GGC GAC AGT GCTATG AGG GCC TTC CAG AAC ACG GCA 144 Lys Val Leu Gly Gly Asp Ser Ala MetArg Ala Phe Gln Asn Thr Ala 35 40 45 ACT GCA TGT GCA CCA GTA TCA CAT TATCGA GCT GTT GAA AGT GTG GAT 192 Thr Ala Cys Ala Pro Val Ser His Tyr ArgAla Val Glu Ser Val Asp 50 55 60 TCA AGT GAA GAA AGT TTT TCT GAT TCA GATGAT GAT AGC TGT CTT TGG 240 Ser Ser Glu Glu Ser Phe Ser Asp Ser Asp AspAsp Ser Cys Leu Trp 65 70 75 80 AAA CGC AAA CGA CAG AAA TGT TTT AAC CCTCCT CCC AAA CCA GAG CCT 288 Lys Arg Lys Arg Gln Lys Cys Phe Asn Pro ProPro Lys Pro Glu Pro 85 90 95 TTT CAG TTT GGC CAG AGC AGT CAG AAA CCA CCTGTT GCT GGA GGA AAG 336 Phe Gln Phe Gly Gln Ser Ser Gln Lys Pro Pro ValAla Gly Gly Lys 100 105 110 AAG ATT AAC AAC ATA TGG GGT GCT GTG CTG CAGGAA CAG AAT CAA GAT 384 Lys Ile Asn Asn Ile Trp Gly Ala Val Leu Gln GluGln Asn Gln Asp 115 120 125 GCA GTG GCC ACT GAA CTT GGT ATC TTG GGA ATGGAG GGC ACT ATT GAC 432 Ala Val Ala Thr Glu Leu Gly Ile Leu Gly Met GluGly Thr Ile Asp 130 135 140 AGA AGC AGA CAA TCC GAG ACC TAC AAT TAT TTGCTT GCC AAG AAA CTT 480 Arg Ser Arg Gln Ser Glu Thr Tyr Asn Tyr Leu LeuAla Lys Lys Leu 145 150 155 160 AGG AAG GAA TCT CAA GAG CAT ACA AAA GATCTA GAC AAG GAA CTA GAT 528 Arg Lys Glu Ser Gln Glu His Thr Lys Asp LeuAsp Lys Glu Leu Asp 165 170 175 GAA TAT ATG CAT GGT GGC AAA AAA ATG GGATCA AAG GAA GAG GAA AAT 576 Glu Tyr Met His Gly Gly Lys Lys Met Gly SerLys Glu Glu Glu Asn 180 185 190 GGG CAA GGT CAT CTC AAA AGG AAA CGA CCTGTC AAA GAC AGG CTA GGG 624 Gly Gln Gly His Leu Lys Arg Lys Arg Pro ValLys Asp Arg Leu Gly 195 200 205 AAC AGA CCA GAA ATG AAC TAT AAA GGT CGATAC GAG ATC ACA GCG GAA 672 Asn Arg Pro Glu Met Asn Tyr Lys Gly Arg TyrGlu Ile Thr Ala Glu 210 215 220 GAT TCT CAA GAG AAA GTG GCT GAT GAA ATTTCA TTC AGG TTA CAG GAA 720 Asp Ser Gln Glu Lys Val Ala Asp Glu Ile SerPhe Arg Leu Gln Glu 225 230 235 240 CCA AAG AAA GAC CTG ATA GCC CGA GTAGTG AGG ATT ATT GGT AAC AAA 768 Pro Lys Lys Asp Leu Ile Ala Arg Val ValArg Ile Ile Gly Asn Lys 245 250 255 AAG GCA ATT GAA CTT CTG ATG GAA ACCGCT GAA GTT GAA CAA AAT GGT 816 Lys Ala Ile Glu Leu Leu Met Glu Thr AlaGlu Val Glu Gln Asn Gly 260 265 270 GGT CTC TTT ATA ATG AAT GGT AGT CGAAGA AGA ACA CCA GGT GGA GTT 864 Gly Leu Phe Ile Met Asn Gly Ser Arg ArgArg Thr Pro Gly Gly Val 275 280 285 TTT CTG AAT CTC TTG AAA AAC ACT CCTAGT ATC AGC GAG GAA CAA ATT 912 Phe Leu Asn Leu Leu Lys Asn Thr Pro SerIle Ser Glu Glu Gln Ile 290 295 300 AAG GAC ATT TTC TAC ATT GAA AAC CAAAAG GAA TAT GAA AAT AAA AAA 960 Lys Asp Ile Phe Tyr Ile Glu Asn Gln LysGlu Tyr Glu Asn Lys Lys 305 310 315 320 GCT GCT AGG AAG AGG AGA ACA CAAGTG TTG GGG AAA AAG ATG AAA CAA 1008 Ala Ala Arg Lys Arg Arg Thr Gln ValLeu Gly Lys Lys Met Lys Gln 325 330 335 GCT ATT AAA AGT CTA AAT TTT CAAGAA GAT GAT GAT ACA TCA CGA GAA 1056 Ala Ile Lys Ser Leu Asn Phe Gln GluAsp Asp Asp Thr Ser Arg Glu 340 345 350 ACT TTT GCA AGT GAC ACG AAT GAGGCC TTG GCC TCT CTT GAT GAG TCA 1104 Thr Phe Ala Ser Asp Thr Asn Glu AlaLeu Ala Ser Leu Asp Glu Ser 355 360 365 CAG GAA GGA CAT GCA GAA GCC AAGTTG GAG GCA GAG GAA GCC ATT GAA 1152 Gln Glu Gly His Ala Glu Ala Lys LeuGlu Ala Glu Glu Ala Ile Glu 370 375 380 GTT GAT CAT TCT CAT GAT TTG GACATC TTT TAA 1185 Val Asp His Ser His Asp Leu Asp Ile Phe 385 390 638base pairs nucleic acid single linear DNA 11 CACCTGCGCA GGCTTGGCTGCGCCTCTCGC GCCGCACGCT CTGGGGTTCC TCCCTTCTTC 60 CGAGCCTCTC CTCTGGCCGCCGCGCGGGAG AGAGGCCGAG ATG GCA GAT GAG ATT 115 Met Ala Asp Glu Ile 1 5GCC AAG GCT CAG GTC GCT CGG CCT GGT GGC GAC ACG ATC TTT GGG AAG 163 AlaLys Ala Gln Val Ala Arg Pro Gly Gly Asp Thr Ile Phe Gly Lys 10 15 20 ATCATC CGC AAG GAA ATA CCA GCC AAA ATC ATT TTT GAG GAT GAC CGG 211 Ile IleArg Lys Glu Ile Pro Ala Lys Ile Ile Phe Glu Asp Asp Arg 25 30 35 TGC CTTGCT TTC CAT GAC ATT TCC CCT CAA GCA CCA ACA CAT TTT CTG 259 Cys Leu AlaPhe His Asp Ile Ser Pro Gln Ala Pro Thr His Phe Leu 40 45 50 GTG ATA CCCAAG AAA CAT ATA TCC CAG ATT TCT GTG GCA GAA GAT GAT 307 Val Ile Pro LysLys His Ile Ser Gln Ile Ser Val Ala Glu Asp Asp 55 60 65 GAT GAA AGT CTTCTT GGA CAC TTA ATG ATT GTT GGC AAG AAA TGT GCT 355 Asp Glu Ser Leu LeuGly His Leu Met Ile Val Gly Lys Lys Cys Ala 70 75 80 85 GCT GAT CTG GGCCTG AAT AAG GGT TAT CGA ATG GTG GTG AAT GAA GGT 403 Ala Asp Leu Gly LeuAsn Lys Gly Tyr Arg Met Val Val Asn Glu Gly 90 95 100 TCA GAT GGT GGACAG TCT GTC TAT CAC GTT CAT CTC CAT GTT CTT GGA 451 Ser Asp Gly Gly GlnSer Val Tyr His Val His Leu His Val Leu Gly 105 110 115 GGT CGG CAA ATGCAT TGG CCT CCT GGT TAA GCACGTTTTG GGGATAATTT 501 Gly Arg Gln Met HisTrp Pro Pro Gly 120 125 TCTCTTCTTT AGGCAATGAT TAAGTTAGGC AATTTCCAGTATGTTAAGTA ACACCTTATT 561 TTTGCCTGTG TATGGAGAGA TTCAAGAAAT AATTTTAAAACCGCATACAT AATAAAAGAC 621 ATTGTTGCAT GGCTTAT 638 127 amino acids aminoacid linear protein 12 Met Asp Val Phe Lys Lys Gly Phe Ser Ile Ala LysLys Gly Val 5 10 15 Val Gly Ala Val Glu Lys Thr Lys Gln Gly Val Thr GluAla Ala 20 25 30 Glu Lys Thr Lys Glu Gly Val Met Tyr Val Gly Ala Lys ThrLys 35 40 45 Glu Asn Val Val Gln Ser Val Thr Ser Val Ala Glu Lys Thr Lys50 55 60 Glu Gln Ala Asn Ala Val Ser Lys Ala Val Val Ser Ser Val Asn 6570 75 Thr Val Ala Thr Lys Thr Val Glu Glu Ala Glu Asn Ile Ala Val 80 8590 Thr Ser Gly Val Val Arg Lys Glu Asp Leu Arg Pro Ser Ala Pro 95 100105 Gln Gln Glu Gly Glu Ala Ser Lys Glu Lys Glu Glu Val Ala Glu 110 115120 Glu Ala Gln Ser Gly Gly Asp 125 443 amino acids amino acid linearprotein 13 Met Asp Arg Leu Gly Ser Phe Ser Asn Asp Pro Ser Asp Lys Pro 510 15 Pro Cys Arg Gly Cys Ser Ser Tyr Leu Met Glu Pro Tyr Ile Lys 20 2530 Cys Ala Glu Cys Gly Pro Pro Pro Phe Phe Leu Cys Leu Gln Cys 35 40 45Phe Thr Arg Gly Phe Glu Tyr Lys Lys His Gln Ser Asp His Thr 50 55 60 TyrGlu Ile Met Thr Ser Asp Phe Pro Val Leu Asp Pro Ser Trp 65 70 75 Thr AlaGln Glu Glu Met Ala Leu Leu Glu Ala Val Met Asp Cys 80 85 90 Gly Phe GlyAsn Trp Gln Asp Val Ala Asn Gln Met Cys Thr Lys 95 100 105 Thr Lys GluGlu Cys Glu Lys His Tyr Met Lys His Phe Ile Asn 110 115 120 Asn Pro LeuPhe Ala Ser Thr Leu Leu Asn Leu Lys Gln Ala Glu 125 130 135 Glu Ala LysThr Ala Asp Thr Ala Ile Pro Phe His Ser Thr Asp 140 145 150 Asp Pro ProArg Pro Thr Phe Asp Ser Leu Leu Ser Arg Asp Met 155 160 165 Ala Gly TyrMet Pro Ala Arg Ala Asp Phe Ile Glu Glu Phe Asp 170 175 180 Asn Tyr AlaGlu Trp Asp Leu Arg Asp Ile Asp Phe Val Glu Asp 185 190 195 Asp Ser AspIle Leu His Ala Leu Lys Met Ala Val Val Asp Ile 200 205 210 Tyr His SerArg Leu Lys Glu Arg Gln Arg Arg Lys Lys Ile Ile 215 220 225 Arg Asp HisGly Leu Ile Asn Leu Arg Lys Phe Gln Leu Met Glu 230 235 240 Arg Arg TyrPro Lys Glu Val Gln Asp Leu Tyr Glu Thr Met Arg 245 250 255 Arg Phe AlaArg Ile Val Gly Pro Val Glu His Asp Lys Phe Ile 260 265 270 Glu Ser HisAla Leu Glu Phe Glu Leu Arg Arg Glu Ile Lys Arg 275 280 285 Leu Gln GluTyr Arg Thr Ala Gly Ile Thr Asn Phe Cys Ser Ala 290 295 300 Arg Thr TyrAsp His Leu Lys Lys Thr Arg Glu Glu Glu Arg Leu 305 310 315 Lys Arg ThrMet Leu Ser Glu Val Leu Gln Tyr Ile Gln Asp Ser 320 325 330 Ser Ala CysGln Gln Trp Leu Arg Arg Gln Ala Asp Ile Asp Ser 335 340 345 Gly Leu SerPro Ser Ile Pro Met Ala Ser Asn Ser Gly Arg Arg 350 355 360 Ser Ala ProPro Leu Asn Leu Thr Gly Leu Pro Gly Thr Glu Lys 365 370 375 Leu Asn GluLys Glu Lys Glu Leu Cys Gln Met Val Arg Leu Val 380 385 390 Pro Gly AlaTyr Leu Glu Tyr Lys Ser Ala Leu Leu Asn Glu Cys 395 400 405 Asn Lys GlnGly Gly Leu Arg Leu Ala Gln Ala Arg Ala Leu Ile 410 415 420 Lys Ile AspVal Asn Lys Thr Arg Lys Ile Tyr Asp Phe Leu Ile 425 430 435 Arg Glu GlyTyr Ile Thr Lys Gly 440 186 amino acids amino acid linear protein 14 MetAsp Ala Asp Ser Asp Val Ala Leu Asp Ile Leu Ile Thr Asn 5 10 15 Val ValCys Val Phe Arg Thr Arg Cys His Leu Asn Leu Arg Lys 20 25 30 Ile Ala LeuGlu Gly Ala Asn Val Ile Tyr Lys Arg Asp Val Gly 35 40 45 Lys Val Leu MetLys Leu Arg Lys Pro Arg Ile Thr Ala Thr Ile 50 55 60 Trp Ser Ser Gly LysIle Ile Cys Thr Gly Ala Thr Ser Glu Glu 65 70 75 Glu Ala Lys Phe Gly AlaArg Arg Leu Ala Arg Ser Leu Gln Lys 80 85 90 Leu Gly Phe Gln Val Ile PheThr Asp Phe Lys Val Val Asn Val 95 100 105 Leu Ala Val Cys Asn Met ProPhe Glu Ile Arg Leu Pro Glu Phe 110 115 120 Thr Lys Asn Asn Arg Pro HisAla Ser Tyr Glu Pro Glu Leu His 125 130 135 Pro Ala Val Cys Tyr Arg IleLys Ser Leu Arg Ala Thr Leu Gln 140 145 150 Ile Phe Ser Thr Gly Ser IleThr Val Thr Gly Pro Asn Val Lys 155 160 165 Ala Val Ala Thr Ala Val GluGln Ile Tyr Pro Phe Val Phe Glu 170 175 180 Ser Arg Lys Glu Ile Leu 185117 amino acids amino acid linear protein 15 Met Asn Ala Pro Pro Ala PheGlu Ser Phe Leu Leu Phe Glu Gly 5 10 15 Glu Lys Lys Ile Thr Ile Asn LysAsp Thr Lys Val Pro Asn Ala 20 25 30 Cys Leu Phe Thr Ile Asn Lys Glu AspHis Thr Leu Gly Asn Ile 35 40 45 Ile Lys Ser Gln Leu Leu Lys Asp Pro GlnVal Leu Phe Ala Gly 50 55 60 Tyr Lys Val Pro His Pro Leu Glu His Lys IleIle Ile Arg Val 65 70 75 Gln Thr Thr Pro Asp Tyr Ser Pro Gln Glu Ala PheThr Asn Ala 80 85 90 Ile Thr Asp Leu Ile Ser Glu Leu Ser Leu Leu Glu GluArg Phe 95 100 105 Arg Val Ala Ile Lys Asp Lys Gln Glu Gly Ile Glu 110115 427 amino acids amino acid linear protein 16 Met Gly Thr Pro Lys ProArg Xaa Leu Pro Trp Leu Val Ser Gln 5 10 15 Leu Asp Leu Gly Gln Leu GluGly Val Ala Trp Val Asn Lys Ser 20 25 30 Arg Thr Arg Phe Arg Ile Pro TrpLys His Gly Leu Arg Gln Asp 35 40 45 Ala Gln Gln Glu Asp Phe Gly Ile PheGln Ala Trp Ala Glu Ala 50 55 60 Thr Gly Ala Tyr Val Pro Gly Arg Asp LysPro Asp Leu Pro Thr 65 70 75 Trp Lys Arg Asn Phe Arg Ser Ala Leu Asn ArgLys Glu Gly Leu 80 85 90 Arg Leu Ala Glu Asp Arg Ser Lys Asp Pro His AspPro His Lys 95 100 105 Ile Tyr Glu Phe Val Asn Ser Gly Val Gly Asp PheSer Gln Pro 110 115 120 Asp Thr Ser Pro Asp Thr Asn Gly Gly Gly Ser ThrSer Asp Thr 125 130 135 Gln Glu Asp Ile Leu Asp Glu Leu Leu Gly Asn MetVal Leu Ala 140 145 150 Pro Leu Pro Asp Pro Gly Pro Pro Ser Leu Ala ValAla Pro Glu 155 160 165 Pro Cys Pro Gln Pro Leu Arg Ser Pro Ser Leu AspAsn Pro Thr 170 175 180 Pro Phe Pro Asn Leu Gly Pro Ser Glu Asn Pro LeuLys Arg Leu 185 190 195 Leu Val Pro Gly Glu Glu Trp Glu Phe Glu Val ThrAla Phe Tyr 200 205 210 Arg Gly Arg Gln Val Phe Gln Gln Thr Ile Ser CysPro Glu Gly 215 220 225 Leu Arg Leu Val Gly Ser Glu Val Gly Asp Arg ThrLeu Pro Gly 230 235 240 Trp Pro Val Thr Leu Pro Asp Pro Gly Met Ser LeuThr Asp Arg 245 250 255 Gly Val Met Ser Tyr Val Arg His Val Leu Ser CysLeu Gly Gly 260 265 270 Gly Leu Ala Leu Trp Arg Ala Gly Gln Trp Leu TrpAla Gln Arg 275 280 285 Leu Gly His Cys His Thr Tyr Trp Ala Val Ser GluGlu Leu Leu 290 295 300 Pro Asn Ser Gly His Gly Pro Asp Gly Glu Val ProLys Asp Lys 305 310 315 Glu Gly Gly Val Phe Asp Leu Gly Pro Phe Ile ValAsp Leu Ile 320 325 330 Thr Phe Thr Glu Gly Ser Gly Arg Ser Pro Arg TyrAla Leu Trp 335 340 345 Phe Cys Val Gly Glu Ser Trp Pro Gln Asp Gln ProTrp Thr Lys 350 355 360 Arg Leu Val Met Val Lys Val Val Pro Thr Cys LeuArg Ala Leu 365 370 375 Val Glu Met Ala Arg Val Gly Gly Ala Ser Ser LeuGlu Asn Thr 380 385 390 Val Asp Leu His Ile Ser Asn Ser His Pro Leu SerLeu Thr Ser 395 400 405 Asp Gln Tyr Lys Ala Tyr Leu Gln Asp Leu Val GluGly Met Asp 410 415 420 Phe Gln Gly Pro Gly Glu Ser 425 252 amino acidsamino acid linear protein 17 Met Gly Gln Cys Gly Ile Thr Ser Ser Lys ThrVal Leu Val Phe 5 10 15 Leu Asn Leu Ile Phe Trp Gly Ala Ala Gly Ile LeuCys Tyr Val 20 25 30 Gly Ala Tyr Val Phe Ile Thr Tyr Asp Asp Tyr Asp HisPhe Phe 35 40 45 Glu Asp Val Tyr Thr Leu Ile Pro Ala Val Val Ile Ile AlaVal 50 55 60 Gly Ala Leu Leu Phe Ile Ile Gly Leu Ile Gly Cys Cys Ala Thr65 70 75 Ile Arg Glu Ser Arg Cys Gly Leu Ala Thr Phe Val Ile Ile Leu 8085 90 Leu Leu Val Phe Val Thr Glu Val Val Val Val Val Leu Gly Tyr 95 100105 Val Tyr Arg Ala Lys Val Glu Asn Glu Val Asp Arg Ser Ile Gln 110 115120 Lys Val Tyr Lys Thr Tyr Asn Gly Thr Asn Pro Asp Ala Ala Ser 125 130135 Arg Ala Ile Asp Tyr Val Gln Arg Gln Leu His Cys Cys Gly Ile 140 145150 His Asn Tyr Ser Asp Trp Glu Asn Thr Asp Trp Phe Lys Glu Thr 155 160165 Lys Asn Gln Ser Val Pro Leu Ser Cys Cys Arg Glu Thr Ala Ser 170 175180 Asn Cys Asn Gly Ser Trp Pro Pro Phe Arg Leu Tyr Ala Glu Gly 185 190195 Cys Glu Ala Leu Val Val Lys Lys Leu Gln Glu Ile Met Met His 200 205210 Val Ile Trp Ala Ala Leu Ala Phe Ala Ala Ile Gln Leu Leu Gly 215 220225 Met Leu Cys Ala Cys Ile Val Leu Cys Arg Arg Ser Arg Asp Pro 230 235240 Ala Tyr Glu Leu Leu Ile Thr Gly Gly Thr Tyr Ala 245 250 417 aminoacids amino acid linear protein 18 Met Ala Ser Ser Ser Gly Ser Ser ProArg Pro Ala Pro Asp Glu 5 10 15 Asn Glu Phe Pro Phe Gly Cys Pro Pro ThrVal Cys Gln Asp Pro 20 25 30 Lys Glu Pro Arg Ala Leu Cys Cys Ala Gly CysLeu Ser Glu Asn 35 40 45 Pro Arg Asn Gly Glu Asp Gln Ile Cys Pro Lys CysArg Gly Glu 50 55 60 Asp Leu Gln Ser Ile Ser Pro Gly Ser Arg Leu Arg ThrGln Glu 65 70 75 Lys Val Gln Ala Glu Val Ala Glu Ala Gly Ile Gly Cys ProPhe 80 85 90 Ala Val Val Gly Cys Ser Phe Lys Gly Ser Pro Gln Phe Val Glu95 100 105 Glu His Glu Val Thr Ser Gln Thr Ser His Leu Asn Leu Leu Leu110 115 120 Gly Phe Met Lys Gln Trp Lys Ala Arg Leu Gly Cys Gly Leu Asp125 130 135 Ser Gly Pro Met Ala Leu Glu Gln Asn Leu Ser Asp Leu Gln Leu140 145 150 Gln Ala Ala Val Glu Val Ala Gly Asp Leu Glu Val Asp Cys Tyr155 160 165 Arg Ala Pro Cys Ser Glu Ser Gln Glu Glu Leu Ala Leu Gln His170 175 180 Phe Met Lys Glu Lys Leu Leu Ala Glu Leu Glu Gly Lys Leu Arg185 190 195 Val Phe Glu Asn Asn Val Ala Val Leu Asn Lys Glu Val Glu Ala200 205 210 Ser His Leu Ala Leu Ala Thr Ser Ile His Gln Ser Gln Leu Asp215 220 225 Arg Glu Arg Ile Leu Ser Leu Glu Gln Arg Val Val Gln Val Gln230 235 240 Gln Thr Leu Ala Gln Lys Asp Gln Ala Leu Gly Lys Leu Glu Gln245 250 255 Ser Leu Arg Leu Met Glu Glu Ala Ser Phe Asp Gly Thr Phe Leu260 265 270 Trp Lys Ile Thr Ser Val Thr Arg Arg Cys His Glu Ser Ala Cys275 280 285 Gly Arg Thr Val Ser Leu Phe Ser Pro Ala Phe Tyr Thr Ala Lys290 295 300 Tyr Gly Tyr Lys Leu Cys Leu Arg Leu Tyr Leu Ile Gly Asp Gly305 310 315 Thr Gly Lys Arg Thr His Leu Ser Leu Phe Ile Val Ile Met Arg320 325 330 Gly Glu Tyr Asp Ala Leu Leu Pro Trp Pro Phe Arg Asn Lys Val335 340 345 Thr Phe Met Leu Leu Asp Gln Asn Asn Arg Glu His Ala Ile Asp350 355 360 Ala Phe Arg Pro Asp Leu Ser Ser Ala Ser Phe Gln Arg Pro Gln365 370 375 Ser Glu Thr Asn Val Ala Ser Gly Cys Pro Leu Phe Phe Pro Leu380 385 390 Ser Lys Leu Gln Ser Pro Lys His Ala Tyr Val Arg Thr Thr Gln395 400 405 Cys Ser Ser Ser Ala Leu Trp Arg Pro Ala Leu Arg 410 415 236amino acids amino acid linear protein 19 Met Ala Arg Ala Cys Leu Gln AlaVal Lys Tyr Leu Met Phe Ala 5 10 15 Phe Asn Leu Leu Phe Trp Leu Gly GlyCys Gly Val Leu Gly Val 20 25 30 Gly Ile Trp Leu Ala Ala Gln Gln Gly SerPhe Ala Thr Leu Ser 35 40 45 Ser Ser Phe Pro Ser Leu Trp Ala Ala Asn LeuLeu Ile Ile Thr 50 55 60 Gly Ala Phe Val Met Ala Ile Gly Phe Val Gly CysLeu Gly Ala 65 70 75 Ile Lys Glu Asn Lys Cys Leu Leu Leu Thr Phe Phe LeuLeu Leu 80 85 90 Leu Leu Val Phe Leu Leu Glu Gly Thr Ile Ala Ile Leu PhePhe 95 100 105 Ala Tyr Thr Asp Lys Ile Asp Arg Tyr Ala Gln Gln Asp LeuLys 110 115 120 Lys Gly Leu His Leu Tyr Gly Thr Gln Gly Asn Val Gly LeuThr 125 130 135 Asn Ala Trp Ser Ile Ile Gln Thr Asp Phe Arg Cys Cys GlyVal 140 145 150 Ser Asn Tyr Thr Asp Trp Phe Glu Val Tyr Asn Ala Thr ArgVal 155 160 165 Pro Asp Ser Cys Cys Leu Glu Phe Ser Glu Ser Cys Gly LeuHis 170 175 180 Ala Pro Ala Leu Val Glu Gly Arg Ala Thr Arg Gly Glu GlyVal 185 190 195 Ala Ser Gly Glu Leu Leu Ala Val Gly Ile Phe Gly Leu CysThr 200 205 210 Ala Leu Val Gln Ile Leu Gly Leu Asn Phe Ala Met Thr MetTyr 215 220 225 Trp Gln Val Val Lys Ala Asp Thr Tyr Cys Ala 230 235 340amino acids amino acid linear protein 20 Met Tyr Gly Asn Tyr Ser His PheMet Lys Phe Pro Ala Gly Tyr 5 10 15 Gly Gly Ser Pro Gly His Thr Gly SerThr Ser Met Ser Pro Ser 20 25 30 Ala Ala Leu Ser Thr Gly Lys Pro Met AspSer His Pro Ser Tyr 35 40 45 Thr Asp Thr Pro Val Ser Ala Pro Arg Thr LeuSer Ala Val Gly 50 55 60 Thr Pro Leu Asn Ala Leu Gly Ser Pro Tyr Arg ValIle Thr Ser 65 70 75 Ala Met Gly Pro Pro Ser Gly Ala Leu Ala Ala Pro ProGly Ile 80 85 90 Asn Leu Val Ala Pro Pro Ser Ser Gln Leu Asn Val Val AsnSer 95 100 105 Val Ser Ser Ser Glu Asp Ile Lys Pro Leu Pro Gly Leu ProGly 110 115 120 Ile Gly Asn Met Asn Tyr Pro Ser Thr Ser Pro Gly Ser LeuVal 125 130 135 Lys His Ile Cys Ala Ile Cys Gly Asp Arg Ser Ser Gly LysHis 140 145 150 Tyr Gly Val Tyr Ser Cys Glu Gly Cys Lys Gly Phe Phe LysArg 155 160 165 Thr Ile Arg Lys Asp Leu Ile Tyr Thr Cys Arg Asp Asn LysAsp 170 175 180 Cys Leu Ile Asp Lys Arg Gln Arg Asn Arg Cys Gln Tyr CysArg 185 190 195 Tyr Gln Lys Cys Leu Val Met Gly Met Lys Arg Glu Ala CysAla 200 205 210 Lys Lys Glu Arg Gln Arg Ser Arg Glu Arg Ala Glu Ser GluAla 215 220 225 Glu Cys Ala Thr Ser Gly His Glu Asp Met Pro Val Glu ArgIle 230 235 240 Leu Glu Ala Glu Leu Ala Val Asp Pro Lys Thr Glu Ser TyrGly 245 250 255 Asp Met Asn Met Glu Asn Ser Thr Asn Asp Pro Val Thr AsnIle 260 265 270 Cys His Ala Ala Asp Lys Gln Leu His Thr Leu Gly Glu TrpAla 275 280 285 Lys Arg Ile Pro His Phe Ser Asp Leu Thr Leu Glu Asp GlnVal 290 295 300 Ile Val Leu Arg Thr Gly Trp Asn Glu Leu Leu Ile Ala SerPhe 305 310 315 Ser His Arg Ser Val Ser Val Glu Asp Gly Ile Pro Leu AlaThr 320 325 330 Gly Leu His Val His Arg Ser Ser Ala His 335 340 394amino acids amino acid linear protein 21 Met Ala Leu Glu Val Gly Asp MetGlu Asp Gly Gln Leu Ser Asp 5 10 15 Ser Asp Ser Asp Met Thr Val Ala ProSer Asp Arg Pro Leu Gln 20 25 30 Leu Pro Lys Val Leu Gly Gly Asp Ser AlaMet Arg Ala Phe Gln 35 40 45 Asn Thr Ala Thr Ala Cys Ala Pro Val Ser HisTyr Arg Ala Val 50 55 60 Glu Ser Val Asp Ser Ser Glu Glu Ser Phe Ser AspSer Asp Asp 65 70 75 Asp Ser Cys Leu Trp Lys Arg Lys Arg Gln Lys Cys PheAsn Pro 80 85 90 Pro Pro Lys Pro Glu Pro Phe Gln Phe Gly Gln Ser Ser GlnLys 95 100 105 Pro Pro Val Ala Gly Gly Lys Lys Ile Asn Asn Ile Trp GlyAla 110 115 120 Val Leu Gln Glu Gln Asn Gln Asp Ala Val Ala Thr Glu LeuGly 125 130 135 Ile Leu Gly Met Glu Gly Thr Ile Asp Arg Ser Arg Gln SerGlu 140 145 150 Thr Tyr Asn Tyr Leu Leu Ala Lys Lys Leu Arg Lys Glu SerGln 155 160 165 Glu His Thr Lys Asp Leu Asp Lys Glu Leu Asp Glu Tyr MetHis 170 175 180 Gly Gly Lys Lys Met Gly Ser Lys Glu Glu Glu Asn Gly GlnGly 185 190 195 His Leu Lys Arg Lys Arg Pro Val Lys Asp Arg Leu Gly AsnArg 200 205 210 Pro Glu Met Asn Tyr Lys Gly Arg Tyr Glu Ile Thr Ala GluAsp 215 220 225 Ser Gln Glu Lys Val Ala Asp Glu Ile Ser Phe Arg Leu GlnGlu 230 235 240 Pro Lys Lys Asp Leu Ile Ala Arg Val Val Arg Ile Ile GlyAsn 245 250 255 Lys Lys Ala Ile Glu Leu Leu Met Glu Thr Ala Glu Val GluGln 260 265 270 Asn Gly Gly Leu Phe Ile Met Asn Gly Ser Arg Arg Arg ThrPro 275 280 285 Gly Gly Val Phe Leu Asn Leu Leu Lys Asn Thr Pro Ser IleSer 290 295 300 Glu Glu Gln Ile Lys Asp Ile Phe Tyr Ile Glu Asn Gln LysGlu 305 310 315 Tyr Glu Asn Lys Lys Ala Ala Arg Lys Arg Arg Thr Gln ValLeu 320 325 330 Gly Lys Lys Met Lys Gln Ala Ile Lys Ser Leu Asn Phe GlnGlu 335 340 345 Asp Asp Asp Thr Ser Arg Glu Thr Phe Ala Ser Asp Thr AsnGlu 350 355 360 Ala Leu Ala Ser Leu Asp Glu Ser Gln Glu Gly His Ala GluAla 365 370 375 Lys Leu Glu Ala Glu Glu Ala Ile Glu Val Asp His Ser HisAsp 380 385 390 Leu Asp Ile Phe 126 AMINO ACIDS AMINO ACID <Unknown>LINEAR PROTEIN 22 Met Ala Asp Glu Ile Ala Lys Ala Gln Val Ala Arg ProGly Gly 5 10 15 Asp Thr Ile Phe Gly Lys Ile Ile Arg Lys Glu Ile Pro AlaLys 20 25 30 Ile Ile Phe Glu Asp Asp Arg Cys Leu Ala Phe His Asp Ile Ser35 40 45 Pro Gln Ala Pro Thr His Phe Leu Val Ile Pro Lys Lys His Ile 5055 60 Ser Gln Ile Ser Val Ala Glu Asp Asp Asp Glu Ser Leu Leu Gly 65 7075 His Leu Met Ile Val Gly Lys Lys Cys Ala Ala Asp Leu Gly Leu 80 85 90Asn Lys Gly Tyr Arg Met Val Val Asn Glu Gly Ser Asp Gly Gly 95 100 105Gln Ser Val Tyr His Val His Leu His Val Leu Gly Gly Arg Gln 110 115 120Met His Trp Pro Pro Gly 125

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide having at least a 95% identity to a member selected fromthe group consisting of: (a) a polynucleotide encoding a polypeptidecomprising the amino acid according to SEQ ID NO:12; (b) apolynucleotide encoding a polypeptide comprising the amino acidaccording to SEQ ID NO:13; (c) a polynucleotide encoding a polypeptidecomprising the amino acid according to SEQ ID NO:14; (d) apolynucleotide encoding a polypeptide comprising the amino acidaccording to SEQ ID NO:15; (e) a polynucleotide encoding a polypeptidecomprising the amino acid according to SEQ ID NO:16; (f) apolynucleotide encoding a polypeptide comprising the amino acidaccording to SEQ ID NO:17; (g) a polynucleotide encoding a polypeptidecomprising the amino acid according to SEQ ID NO:18; (h) apolynucleotide encoding a polypeptide comprising the amino acidaccording to SEQ ID NO:19; (i) a polynucleotide encoding a polypeptidecomprising the amino acid according to SEQ ID NO:20; (j) apolynucleotide encoding a polypeptide comprising the amino acidaccording to SEQ ID NO:21; (k) a polynucleotide encoding a polypeptidecomprising the amino acid according to SEQ ID NO:22; and (l) thecomplement of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), and (k).2. The isolated polynucleotide of claim 1 wherein said member is (a). 3.The isolated polynucleotide of claim 1 wherein said member is (b). 4.The isolated polynucleotide of claim 1 wherein said member is (c). 5.The isolated polynucleotide of claim 1 wherein said member is (d). 6.The isolated polynucleotide of claim 1 wherein said member is (e). 7.The isolated polynucleotide of claim 1 wherein said member is (f). 8.The isolated polynucleotide of claim 1 wherein said member is (g). 9.The isolated polynucleotide of claim 1 wherein said member is (h). 10.The isolated polynucleotide of claim 1 wherein said member is (i). 11.The isolated polynucleotide of claim 1 wherein said member is (j). 12.The isolated polynucleotide of claim 1 wherein said member is (k). 13.The isolated polynucleotide of claim 1, wherein the polynucleotide isDNA.
 14. A method of making a recombinant vector comprising insertingthe isolated polynucleotide of claim 1 into a vector, wherein saidpolynucleotide is DNA.
 15. A recombinant vector comprising thepolynucleotide of claim 1, wherein the polynucleotide is DNA.
 16. Arecombinant host cell comprising the polynucleotide of claim 1, whereinsaid polynucleotide is DNA.
 17. A method for producing a polypeptidecomprising expressing from the recombinant cell of claim 16 thepolypeptide encoded by said polynucleotide.
 18. The isolatedpolynucleotide of claim 1 comprising the nucleotide sequence which is amember selected from the group consisting of: (a) a polynucleotidecomprising the sequence according to SEQ ID NO:1; (b) a polynucleotidecomprising the sequence according to SEQ ID NO:2; (c) a polynucleotidecomprising the sequence according to SEQ ID NO:3; (d) a polynucleotidecomprising the sequence according to SEQ ID NO:4; (e) a polynucleotidecomprising the sequence according to SEQ ID NO:5; (f) a polynucleotidecomprising the sequence according to SEQ ID NO:6; (g) a polynucleotidecomprising the sequence according to SEQ ID NO:7; (h) a polynucleotidecomprising the sequence according to SEQ ID NO:8; (i) a polynucleotidecomprising the sequence according to SEQ ID NO:9; (j) a polynucleotidecomprising the sequence according to SEQ ID NO:10; and (k) apolynucleotide comprising the sequence according to SEQ ID NO:11.
 19. Anisolated polypeptide comprising a polypeptide having at least a 95%identity to a member selected form the group consisting of: (a) apolypeptide comprising the amino acid according to SEQ ID NO:12; (b) apolypeptide comprising the amino acid according to SEQ ID NO:13; (c) apolypeptide comprising the amino acid according to SEQ ID NO:14; (d) apolypeptide comprising the amino acid according to SEQ ID NO:15; (e) apolypeptide comprising the amino acid according to SEQ ID NO:16; (f) apolypeptide comprising the amino acid according to SEQ ID NO:17; (g) apolypeptide comprising the amino acid according to SEQ ID NO:18; (h) apolypeptide comprising the amino acid according to SEQ ID NO:19; (i) apolypeptide comprising the amino acid according to SEQ ID NO:20; (j) apolypeptide comprising the amino acid according to SEQ ID NO:21; and (k)a polypeptide comprising the amino acid according to SEQ ID NO:22. 20.An antibody that specifically binds the polypeptide of claim
 19. 21. Amethod for identifying an antagonist of the polypeptide of claim 19,comprising: (a) contacting a cell expressing said polypeptide with acompound to be screened; and (b) determining whether said compound actsas an antagonist of said polypeptide.
 22. A method for identifying anagonist of the polypeptide of claim 19, comprising: (a) contacting acell expressing said polypeptide with a compound to be screened; and (b)determining whether said compound acts as an agonist of saidpolypeptide.